NOGO Receptor Homologs

ABSTRACT

The invention relates generally to genes that encode proteins that inhibit axonal growth. The invention relates specifically to genes encoding NgR protein homologs in humans and mice. The invention also includes compositions and methods for modulating the expression and activity of Nogo and the NgR proteins. Specifically, the invention includes peptides, proteins and antibodies that block Nogo-mediated inhibition of axonal extension. The compositions and methods of the invention are useful in the treatment of cranial or cerebral trauma, spinal cord injury, stroke or a demyelinating disease.

FIELD OF THE INVENTION

The invention relates to neurology and molecular biology. Moreparticularly, the invention relates to CNS neurons and axonal growth

BACKGROUND

Among the mechanisms through which the cells of an organism communicatewith each other and obtain information and stimuli from theirenvironment is through cell membrane receptor molecules expressed on thecell surface. Many such receptors have been identified, characterized,and sometimes classified into major receptor superfamilies based onstructural motifs and signal transduction features. The receptors are afirst essential link for translating an extracellular signal into acellular physiological response.

Receptors on neurons are particularly important in the development ofthe nervous system during embryogenesis. The neurons form connectionswith target cells during development through axonal extension of theneurons toward the target cells in a receptor-mediated process. Axonsand dendrites have a specialized region of their distal tips known asthe growth cone. Growth cones enable the neuron to sense the localenvironment through a receptor-mediated process and direct the movementof the axon or dendrite of the neuron toward the neuron's target cell.This process is known as elongation. Growth cones can be sensitive toseveral guidance cues, for example, surface adhesiveness growth factors,neurotransmitters and electric fields. The guidance of growth at thecone depends on various classes of adhesion molecules, intercellularsignals, as well as factors that stimulate and inhibit growth cones.

Interestingly, damaged neurons do not elongate in the central nervoussystem (CNS) following injury due to trauma or disease, whereas axons inthe peripheral nervous system (PNS) regenerate readily. The fact thatdamaged CNS neurons fail to elongate is not due to an intrinsic propertyof CNS axons, but rather due to the CNS environment that is notpermissive for axonal elongation. Classical grafting experiments byAguayo and colleagues (e.g., Richardson et al., (1980) Nature 284,264-265) demonstrated that CNS axons can in fact elongate oversubstantial distances within peripheral nerve grafts, and that CNSmyelin inhibits CNS axon elongation. Therefore, given the appropriateenvironment, CNS axons can regenerate, implying that CNS axonal injurycan potentially be addressed by appropriate manipulation of the CNSenvironment.

The absence of axon regeneration following injury can be attributed tothe presence of axon growth inhibitors. These inhibitors arepredominantly associated with myelin and constitute an important barrierto regeneration. Axon growth inhibitors are present in CNS-derivedmyelin and the plasma membrane of oligodendrocytes that synthesizemyelin in the CNS (Schwab et al., (1993) Annu. Rev. Neurosci. 16,565-595). Myelin-associated inhibitors appear to be a primarycontributor to the failure of CNS axon regeneration in vivo after aninterruption of axonal continuity, whereas other non-myelin associatedaxon growth inhibitors in the CNS may play a lesser role. Theseinhibitors block axonal regeneration following neuronal injury due totrauma, stroke or viral infection.

Numerous myelin-derived axon growth inhibitors have been characterized(see, for review, David et al., (1999) WO995394547; Bandman et al.,(1999) U.S. Pat. No. 5,858,708; Schwab, (1996) Neurochem. Res. 21,755-761). Several components of CNS white matter, NI35, NI250 (Nogo) andMyelin-associated glycoprotein (MAG), which have inhibitory activity foraxonal extension, have been described as well (Schwab et al., (1990)WO9005191; Schwab et al., (1997) U.S. Pat. No. 5,684,133). Inparticular, Nogo is a 250 kDa myelin-associated axon growth inhibitorthat was originally characterized based on the effects of the purifiedprotein in vitro and monoclonal antibodies that neutralize the protein'sactivity (Schwab (1990) Exp. Neurol. 109, 2-5). The Nogo cDNA was firstidentified through random analysis of brain cDNA and had no suggestedfunction (Nagase et al., (1998) DNA Res. 5, 355-364). The identificationof this Nogo cDNA as the cDNA encoding the 250 kDa myelin-associatedaxon growth inhibitor was discovered only recently (GrandPre et al.,(2000) Nature 403, 439-444; Chen et al., (2000) Nature 403, 434-439;Prinjha at al., (2000) Nature 403, 383-384).

Importantly, Nogo has been shown to be the primary component of CNSmyelin responsible for inhibiting axonal elongation and regeneration.Nogo's selective expression by oligodendrocytes and not by Schwann cells(the cells that myelinate P.S. axons) is consistent with the inhibitoryeffects of CNS myelin, in contrast to P.S. myelin (GrandPre et al.,(2000) Nature 403, 434-439). In culture, Nogo inhibits axonal elongationand causes growth cone collapse (Spillmann et al., (1998) J. Biol. Chem.272, 19283-19293). Antibodies (e.g., IN-1) against Nogo have been shownto block most of the inhibitory action of CNS myelin on neurite growthin vitro (Spillmann et al., (1998) J. Biol. Chem. 272:19283-19293).These experiments indicate that Nogo is the main component of CNS myelinresponsible for inhibition of axonal elongation in culture. Furthermore,in vivo, the IN-1 antibody has been shown to enhance axonal regenerationafter spinal cord injury, resulting in recovery of behaviors such ascontact placing and stride length (Schnell and Schwab (1990) Nature 343,269-272; Bregman et al., (1995) Nature 378, 498-501). Thus, there issubstantial evidence that Nogo is a disease-relevant molecular target.Agents that interfere with the binding of Nogo to its receptor would beexpected to improve axonal regeneration in clinical states in whichaxons have been damaged, and improve patient outcome.

Modulation of Nogo has been described as a means for treatment ofregeneration for neurons damaged by trauma, infarction and degenerativedisorders of the CNS (Schwab et al., (1994) WO9417831; Tatagiba et al.,(1997) Neurosurgery 40, 541-546) as well as malignant tumors in the CNSsuch as glioblastoma (Schwab et al., (1993) U.S. Pat. No. 5,250,414);Schwab et al., (2000) U.S. Pat. No. 6,025,333).

Antibodies which recognize Nogo have been suggested to be useful in thediagnosis and treatment of nerve damage resulting from trauma,infarction and degenerative disorders of the CNS (Schnell & Schwab,(1990) Nature 343, 269-272, Schwab et al., (1997) U.S. Pat. No.5,684,133). For CNS axons, there is a correlation between the presenceof myelin and the inhibition of axon regeneration over long distances(Savio and Schwab (1990) Proc. Natl. Acad. Sci. 87, 4130-4133; Keirsteadet al., (1992) Proc. Natl. Acad. Sci. 89, 11664-11668). After Nogo isblocked by antibodies, neurons can again extend across lesions caused bynerve damage (Schnell and Schwab (1990) Nature 343, 269-272).

SUMMARY OF THE INVENTION

Genes encoding homologs (NgR2 and NgR3) of a Nogo receptor (NgR1) inmice and humans have been discovered. Various domains in thepolypeptides encoded by the NgR2 and NgR3 genes have been identified andcompared to domains in mouse and human NGR1 polypeptides. Thiscomparison has led to identification of a consensus sequence (NgRconsensus sequence) that characterizes a family of proteins (NgRfamily). Based on these and other discoveries, the invention featuresmolecules and methods for modulating axonal growth in CNS neurons.

The invention provides a polypeptide that contains a polypeptidecontaining a tryptophan rich LRRCT domain consisting of the amino acidsequence:

[SEQ ID NO: 19] N X₁ W X₂ C X₃ C R A R X₄ L W X₅ W X₆ X₇ X₈ X₉ R X₁₀S S S X₁₁ V X₁₂ C X₁₃ X₁₄ P X₁₅ X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ D LX₂₁ X₂₂ L X₂₃ X₂₄ X₂₅ D X₂₆ X₂₇ X₂₈ C

wherein X is any protein amino acid or a gap, and the polypeptide doesnot include amino acid sequence from residue 260 to 309 of SEQ ID NO: 5(human NGR1) or SEQ ID NO: 17 (mouse NgR1).

Preferably, X17 and X23 are (independently) arginine or lysine. In someembodiments, the amino acid sequence of the LRRCT domain is residues261-310 of SEQ ID NO:2, or residues 261-310 of SEQ ID NO. 2 with up to10 conservative amino acid substitutions. In some embodiments, thepolypeptide contains the following NTLRRCT amino acid sequence:

[SEQ ID NO: 18] C P X₁ X₂ C X₃ C Y X₄ X₅ P X₆ X₇ T X₈ S C X₉ X₁₀ X₁₁ X₁₂X₁₃ X₁₄ X₁₅ X₁₆ P X₁₇ X₁₈ X₁₉ P X₂₀ X₂₁ X₂₂ X₂₃ R X₂₄ F LX₂₅ X₂₆ N X₂₇ I X₂₈ X₂₉ X₃₀ X₃₁ X₃₂ X₃₃ X₃₄ F X₃₅ X₃₆ X₃₇X₃₈ X₃₉ X₄₀ X₄₁ X₄₂ L W X₄₃ X₄₄ S N X₄₅ X₄₆ X₄₇ X₄₈ I X₄₉X₅₀ X₅₁ X₅₂ F X₅₃ X₅₄ X₅₅ X₅₆ X₅₇ L E X₅₈ L D L X₅₉ D N X₆₀ X₆₁ L X₆₂ X₆₃ X₆₄ X₆₅ P X₆₆ T F X₆₇ G L X₆₈ X₆₉ L X₇₀X₇₁ L X₇₂ L X₇₃ X₇₄ C X₇₅ L X₇₆ X₇₇ X₇₈ X₇₉ X₈₀ X₈₁ F X₈₂ GL X₈₃ X₈₄ L Q Y L Y L Q X₈₅ N X₈₆ X₈₇ X₈₈ X₈₉ L X₉₀ D X₉₁X₉₂ F X₉₃ D L X₉₄ N L X₉₅ H L F L H G N X₉₆ X₉₇ X₉₈ X₉₉X₁₀₀ X₁₀₁ X₁₀₂ X₁₀₃ X₁₀₄ F R G L X₁₀₅ X₁₀₆ L D R L L L HX₁₀₇ N X₁₀₈ X₁₀₉ X₁₁₀ X₁₁₁ V H X₁₁₂ X₁₁₃ A F X₁₁₄ X₁₁₅ L X₁₁₆R L X₁₁₇ X₁₁₈ L X₁₁₉ L F X₁₂₀ N X₁₂₁ L X₁₂₂ X₁₂₃ L X₁₂₄ X₁₂₅X₁₂₆ X₁₂₇ L X₁₂₈ X₁₂₉ L X₁₃₀ X₁₃₁ L X₁₃₂ X₁₃₃ L R L N X₁₃₄N X₁₃₅ W X₁₃₆ C X₁₃₇ C R X₁₃₈ R X₁₃₉ L W X₁₄₀ W X₁₄₁ X₁₄₂X₁₄₃ X₁₄₄ R X₁₄₅ S S S X₁₄₆ V X₁₄₇ C X₁₄₈ X₁₄₉ P X₁₅₀ X₁₅₁X₁₅₂ X₁₅₃ X₁₅₄ X₁₅₅ D L X₁₅₆ X₁₅₇ L X₁₅₈ X₁₅₉ X₁₆₀ D X₁₆₁ X₁₆₂ X₁₆₃ Cwherein X is any amino acid residue or a gap and wherein the polypeptideis not the polypeptide of SEQ ID NO: 5 (human NGR1) or SEQ ID NO: 17(mouse NgR1). For example, X₆, X₃₇ and X₃₈ may represent a gap. Specificexamples of polypeptides of the invention are SEQ ID NO: 2 (human NgR2),SEQ ID NO: 4 (mouse NgR3), and SEQ ID NO: 14 (human NgR3). In someembodiments, the polypeptide contains: (a) a NTLRRCT domain, and (b)less than a complete CTS domain, provided that a partial CTS domain, ifpresent, consists of no more than the first 39 amino acids of the CTSdomain. While the polypeptide may contain a functional GPI domain, afunctional GPI domain may be absent, e.g., when a soluble polypeptide isdesired. A polypeptide of the invention optionally includes an aminoacid sequence of a heterologous polypeptide, e.g., an Fc portion of anantibody.

The invention also provides a nucleic acid encoding an above-describedpolypeptide; a vector containing the nucleic acid, which nucleic acidmay be operably linked to an expression control sequence; and atransformed host cell containing the vector. A method of producing apolypeptide of the invention is also provided. The method includesintroducing a nucleic acid encoding the above-described polypeptide intoa host cell, culturing the cell under conditions suitable for expressionof the polypeptide, and recovering the polypeptide.

The invention also provides an antisense molecule whose nucleotidesequence is complementary to a nucleotide sequence encoding apolypeptide selected from the group consisting of: a polypeptideconsisting of residues 311-395 of SEQ ID NO: 2, a polypeptide consistingof residues 256-396 of SEQ ID NO: 14 and a polypeptide consisting ofresidues 321-438 of SEQ ID NO: 4, wherein the nucleic acid is from 8 to100 nucleotides in length, e.g., about 20, 30, 40, 50, 60, 70, 80 or 90nucleotides. The invention also provides a nucleic acid encoding such anantisense molecule.

The invention also provides an antibody that binds to an above-describedpolypeptide. Polypeptides or antibodies of the invention can beformulated into pharmaceutical compositions containing the polypeptideor antibody and a pharmaceutically acceptable carrier.

The invention also provides a method for decreasing inhibition of axonalgrowth of a CNS neuron. The method includes the step of contacting theneuron with an effective amount of a polypeptide or antibody of theinvention.

The invention also provides a method for treating a central nervoussystem disease, disorder or injury. The method includes administering toa mammal, e.g., a human, an effective amount of a polypeptide orantibody of the invention. Exemplary diseases, disorders and injuriesthat may be treated using molecules and methods of the inventioninclude, but are not limited to, cerebral injury, spinal cord injury,stroke, demyelinating diseases, e.g., multiple sclerosis, monophasicdemyelination, encephalomyelitis, multifocal leukoencephalopathy,panencephalitis, Marchiafava-Bignami disease, Spongy degeneration,Alexander's disease, Canavan's disease, metachromatic leukodystrophy andKrabbe's disease.

The invention also provides a method for identifying a molecule thatbinds a polypeptide of the invention. The method includes the steps of:(a) providing a polypeptide of the invention; (b) contacting thepolypeptide with the candidate molecule; and (c) detecting binding ofthe candidate molecule to the polypeptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. In case of conflict, the presentapplication, including definitions, will control. All publications,patent and other references mentioned herein are incorporated byreference.

The materials, methods and examples presented below are illustrativeonly, and not intended to be limiting. Other features and advantages ofthe invention will be apparent from the detail description and from theclaims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B shows an alignment of NgR2 (SEQ ID NO:2) and NgR3 (SEQ IDNO:4) with the known NgR, NgR1 (SEQ ID NO:5) and the Consensus Sequence(SEQ ID NO:6).

FIG. 2. mNgR3 does not bind hNogoA(1055-1120). COS-7 cells weretransfected with vectors encoding myc-NgR1 or myc-NgR3, fixed, andstained with anti-myc antibodies or AP-hNogoA(1055-1120).

FIG. 3. An alignment of the amino acid sequences of human NgR1, murineNGR1, murine NgR3, human NgR3 and human NgR2. Numbering begins withamino acid #1 of murine NgR3. The consensus sequence is listed below.The LRR NT domain is indicated by a shaded box; domains LLR 1, LLR 3,LLR 5, and LLR 7 are indicated by open boxes; LLR 2, LLR 4, LLR 6 andLLR 8 are indicated by shaded boxes; and the LLR CT domain is indicatedby a shaded box. Amino acids in bold in LLR 8 indicate a conservedglycosylation sites. A dot indicates conserved cystine residue in LRR4.Box at C terminus indicates putative GPI signals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides purified and isolated polynucleotides(e.g., DNA sequences and RNA transcripts, both sense and complementaryantisense strands, both single- and double-stranded, including splicevariants thereof) encoding NgR homologs, referred to herein as NgR.Unless indicated otherwise, as used herein, the abbreviation in lowercase (NgR) refers to a gene, cDNA, RNA or nucleic acid sequence, whereasthe upper case version (NgR) refers to a protein, polypeptide, peptide,oligopeptide, or amino acid sequence. Specific proteins are designatedby number, e.g., “NgR2” is a human NgR homolog, “NgR3” is amurine-derived NgR homolog, and “NgR1” is the known NgR identified byDr. Stephen Strittmatter. Known NgRs are herein referred to as “NgRs.”DNA polynucleotides of the invention include genomic DNA, cDNA and DNAthat has been chemically synthesized in whole or in part.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York (1998); Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).

As used herein, the term “axon” refers to a long cellular protrusionfrom a neuron, whereby action potentials are conducted, either to orfrom the cell body.

As used herein, the term “axonal growth” refers to an extension of thelong process or axon, originating at the cell body and proceeded by thegrowth cone.

As used herein, the term “central nervous system disorder” refers to anypathological state associated with abnormal function of the centralnervous system (CNS). The term includes, but is not limited to, alteredCNS function resulting from physical trauma to cerebral tissue, viralinfection, autoimmune mechanisms and genetic mutation.

As used herein, the term “demyelinating disease” refers to apathological disorder characterized by the degradation of the myelinsheath of the oligodendrocyte cell membrane.

As used herein, the term “growth cone” refers to a specialized region atthe tip of a growing neurite that is responsible for sensing the localenvironment and moving the axon toward its appropriate synaptic targetcell.

As used herein, the term “growth cone movement” refers to the extensionor collapse of the growth cone toward a neuron's target cell.

As used herein, the term “neurite” refers to a process growing out of aneuron. As it is sometimes difficult to distinguish a dendrite from inaxon in culture, the term “neurite” is used for both.

As used herein, the term “oligodendrocyte” refers to a neuroglial cellof the CNS whose function is to myelinate CNS axons.

“Synthesized” as used herein and understood in the art, refers topolynucleotides produced by purely chemical, as opposed to enzymatic,methods. “Wholly” synthesized DNA sequences are therefore producedentirely by chemical means, and “partially” synthesized DNAs embracethose wherein only portions of the resulting DNA were produced bychemical means. By the term “region” is meant a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein. The term “domain” is herein defined asreferring to a structural part of a biomolecule that contributes to aknown or suspected function of the biomolecule. Domains may beco-extensive with regions or portions thereof; domains may alsoincorporate a portion of a biomolecule that is distinct from aparticular region, in addition to all or part of that region. Examplesof NgR protein domains include, but are not limited to, the signalpeptide, extracellular (i.e., N-terminal) domain, and leucine-richrepeat domains.

As used herein, the term “activity” refers to a variety of measurableindicia suggesting or revealing binding, either direct or indirect;affecting a response, i.e., having a measurable affect in response tosome exposure or stimulus, including, for example, the affinity of acompound for directly binding a polypeptide or polynucleotide of theinvention, or, for example, measurement of amounts of upstream ordownstream proteins or other similar functions after some stimulus orevent. Such activities may be measured by assays such as competitiveinhibition of NGR1 binding to Nogo assays wherein, for example,unlabeled, soluble NgR2 is added to an assay system in increasingconcentrations to inhibit the binding of Nogo to NGR1 expressed on thesurface of CHO cells. As another example, one may assess the ability ofneurons to extend across lesions caused by nerve damage (as in Schnelland Schwab (1990) Nature 343, 269-272) following inhibition of Nogo byvarious forms of NgR2 and/or NgR3 as a biological indicator of NgRfunction.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab, Fab′, F(ab)2, and other fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies,anti-anti-idiotypic antibodies, and humanized antibodies.

As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof. Binding includes ionic, non-ionic,hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Thephysical interaction, the binding, can be either direct or indirect,indirect being through or due to the effects of another protein orcompound. Direct binding refers to interactions that do not take placethrough or due to the effect of another protein or compound but insteadare without other substantial chemical intermediates.

As used herein, the term “compound” means any identifiable chemical ormolecule, including, but not limited to, small molecules, peptides,proteins, sugars, nucleotides or nucleic acids, and such compound can benatural or synthetic.

As used herein, the term “complementary” refers to Watson-Crickbasepairing between nucleotide units of a nucleic acid molecule.

As used herein, the term “contacting” means bringing together, eitherdirectly or indirectly, a compound into physical proximity to apolypeptide or polynucleotide of the invention. The polypeptide orpolynucleotide can be in any number of buffers, salts, solutions etc.Contacting includes, for example, placing the compound into a beaker,microtiter plate, cell culture flask, or a microarray, such as a genechip, or the like, which contains the nucleic acid molecule, orpolypeptide encoding the NgR or fragment thereof.

As used herein, the phrase “homologous nucleotide sequence,” or“homologous amino acid sequence,” or variations thereof, refers tosequences characterized by an identity at the nucleotide level, or ahomology at the amino acid level, of at least the specified percentage.Homologous nucleotide sequences include those sequences coding forisoforms of proteins. Such isoforms can be expressed in differenttissues of the same organism as a result of, for example, alternativesplicing of RNA. Alternatively, isoforms can be encoded by differentgenes. Homologous nucleotide sequences include nucleotide sequencesencoding for a protein of a species other than humans, including, butnot limited to, mammals. Homologous nucleotide sequences also include,but are not limited to, naturally occurring allelic variations andmutations of the nucleotide sequences set forth herein. A homologousnucleotide sequence does not, however, include the nucleotide sequenceencoding NgR1. Homologous amino acid sequences include those amino acidsequences which contain conservative amino acid substitutions and whichpolypeptides have the same binding and/or activity. A homologous aminoacid sequence does not, however, include the amino acid sequenceencoding other known NgRs. Percent homology can be determined by, forexample, the Gap program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, MadisonWis.), using the default settings, which uses the algorithm of Smith andWaterman (Adv. Appl. Math, 1981, 2, 482-489, which is incorporatedherein by reference in its entirety).

As used herein, the term “isolated” nucleic acid molecule refers to anucleic acid molecule (DNA or RNA) that is substantially free of nucleicacids encoding other proteins with which it is associated in nature,i.e., a nucleic acid that has been removed from its native environment.Examples of isolated nucleic acid molecules include, but are not limitedto, recombinant DNA molecules contained in a vector, recombinant DNAmolecules maintained in a heterologous host cell, partially orsubstantially purified nucleic acid molecules, and synthetic DNA or RNAmolecules. Preferably, an “isolated” nucleic acid is free of sequenceswhich naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic. DNA of the organismfrom which the nucleic acid is derived. For example, in variousembodiments, the isolated NgR nucleic acid molecule can contain lessthan about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleotide sequences which naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material or culture mediumwhen produced by recombinant techniques, or of chemical precursors orother chemicals when chemically synthesized.

As used herein, the term “heterologous” refers to a nucleotide or aminoacid sequence that is a different, or non-corresponding sequence, or asequence derived from a different species. For example, a mouse NgRnucleotide or amino acid sequence is heterologous to a human NgRnucleotide or amino acid sequence, and a human NgR nucleic or amino acidsequence is heterologous to a human immunoglobulin nucleotide or aminoacid sequence.

As used herein, a “soluble NgR polypeptide” is a NgR polypeptide thatdoes not anchor itself in a membrane. Such soluble polypeptides include,for example, NgR2 and NgR3 polypeptides that lack a sufficient portionof their GPI anchor signal to anchor the polypeptide or are modifiedsuch that the GPI anchor signal is not adequate to result in replacementof the peptide with a GPI anchor. In preferred embodiments, up to 5, 10,20 or 25 amino acids are removed from the C-terminus of NgR2 or NgR3 tomake the respective proteins soluble. As used herein soluble NgRpolypeptides include full-length or truncated (e.g., with internaldeletions) NgR.

Soluble NgR polypeptides may include the entire NgR protein up to theputative GPI signal sequence (e.g., amino acid 1 to about amino acid 395of NgR2, and from amino acid 1 to about amino acid 438 of NgR3). Inother embodiments, the signal peptide of the proteins may be removed ortruncated (e.g., all or part of the signal sequence of NgR2, which spansamino acid 1 to about amino acid 30 of SEQ ID NO:2, may be removed; allor part of the signal sequence of NgR3, which spans amino acid 1 toabout amino acid 40 of SEQ ID NO:4, may be removed). In someembodiments, the mature NgR2 (SEQ ID NO:8) and the mature NgR3 (SEQ IDNO:9) are used.

Soluble NgR polypeptides include at least one of the putativeligand-binding portions of NgR, including the first cysteine-rich region(SEQ ID NO:10, the leucine repeat region (SEQ ID NO:12) and the secondcysteine-rich region (SEQ ID NO:11). In some embodiments, soluble NgRpolypeptides consist of amino acid 1 through about amino acid 395 of SEQID NO:2, or amino acid 1 through about amino acid 438 of SEQ ID NO:4.

In other embodiments, the soluble NgR polypeptides are fusion proteinsthat contain amino acids 30 through about amino acid 395 of mature NgR2or amino acid 40 through about amino acid 438 of NgR3, the C-terminal 10amino acids of a human IgG 1 hinge region containing the two cysteineresidues thought to participate in interchain disulfide bonding, and theCH2 and CH3 regions of a human IgGI heavy chain constant domain. Thistype of recombinant protein is designed to modulate inhibition of axonalelongation through inhibition of the Nogo ligand binding to NGR1, or byinhibiting the ligand of the NgR from interacting with cell surface NgRThe NgR portion of the fusion binds to the Nogo ligand and the IgG1portion binds to the FcγRI (macrophage) and FcγIII (NK cells andneutrophils) receptors.

The production of the soluble polypeptides useful in this invention maybe achieved by a variety of methods known in the art. For example, thepolypeptides may be derived from intact transmembrane NgR molecules byproteolysis using specific endopeptidases in combination withexopeptidases, Edman degradation, or both. The intact NgR molecule, inturn, may be purified from its natural source using conventionalmethods. Alternatively, the intact NgR may be produced by knownrecombinant DNA techniques using cDNAs, expression vectors andwell-known techniques for recombinant gene expression.

Preferably, the soluble polypeptides useful in the present invention areproduced directly, thus eliminating the need for an entire NgR as astarting material. This may be achieved by conventional chemicalsynthesis techniques or by well-known recombinant DNA techniques whereinonly those DNA sequences which encode the desired peptides are expressedin transformed hosts. For example, a gene which encodes the desiredsoluble NgR polypeptide may be synthesized by chemical means using anoligonucleotide synthesizer. Such oligonucleotides are designed based onthe amino acid sequence of the desired soluble NgR polypeptide. SpecificDNA sequences coding for the desired peptide also can be derived fromthe full-length DNA sequence by isolation of specific restrictionendonuclease fragments or by PCR synthesis of the specified region fromcDNA.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NOs:1, 3 or acomplement of either of these nucleotide sequences, can be isolatedusing standard molecular biology techniques and the sequence informationprovided herein. Using all or a portion of the nucleic acid sequences ofSEQ ID NOs:1 or 3 as a hybridization probe, NgR nucleic acid sequencescan be isolated using standard hybridization and cloning techniques(e.g. as described in Sambrook et al., eds., MOLECULAR CLONING: ALABORATORY MANUAL 2^(nd) Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLSIN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993).

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to NgR nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

As used herein, the terms “modulates” or “modifies” means an increase ordecrease in the amount, quality, or effect of a particular activity orprotein.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues which has a sufficient number of bases to be used ina polymerase chain reaction (PCR). This short sequence is based on (ordesigned from) a genomic or cDNA sequence and is used to amplify,confirm or reveal the presence of an identical, similar or complementaryDNA or RNA in a particular cell or tissue. Oligonucleotides compriseportions of a DNA sequence having at least about 10 nucleotides and asmany as about 50 nucleotides, preferably about 15 to 30 nucleotides.They are chemically synthesized and may be used as probes.

As used herein, the term “probe” refers to nucleic acid sequences ofvariable length, preferably between at least about 10 and as many asabout 6,000 nucleotides, depending on use. They are used in thedetection of identical, similar or complementary nucleic acid sequences.Longer length probes are usually obtained from a natural or recombinantsource, are highly specific and much slower to hybridize than oligomers.They may be single- or double-stranded and carefully designed to havespecificity in PCR, hybridization membrane-based, or ELISA-liketechnologies.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism.

The term “therapeutic effect” refers to the inhibition or activationfactors causing or contributing to the abnormal condition. A therapeuticeffect relieves to some extent one or more of the symptoms of theabnormal condition. In reference to the treatment of abnormalconditions, a therapeutic effect can refer to one or more of thefollowing: (a) an increase in the proliferation, growth, and/ordifferentiation of cells; (b) inhibition (i.e., slowing or stopping) ofcell death; (c) inhibition of degeneration; (d) relieving to some extentone or more of the symptoms associated with the abnormal condition; and(e) enhancing the function of the affected population of cells.Compounds demonstrating efficacy against abnormal conditions can beidentified as described herein.

The term “abnormal condition” refers to a function in the cells ortissues of an organism that deviates from their normal functions in thatorganism. An abnormal condition can relate to cell proliferation, celldifferentiation, cell signaling, or cell survival. An abnormal conditionmay also include obesity, diabetic complications such as retinaldegeneration, and irregularities in glucose uptake and metabolism, andfatty acid uptake and metabolism.

Abnormal cell proliferative conditions, for example, include cancerssuch as fibrotic and mesangial disorders, abnormal angiogenesis andvasculogenesis, wound healing, psoriasis, diabetes mellitus andinflammation.

Abnormal differentiation conditions include, for example,neurodegenerative disorders, slow wound healing rates and slow tissuegrafting healing rates.

Abnormal cell signaling conditions include, for example, psychiatricdisorders involving excess neurotransmitter activity.

Abnormal cell survival conditions may also relate to conditions in whichprogrammed cell death (apoptosis) pathways are activated or abrogated. Anumber of protein kinases are associated with the apoptosis pathwaysAberrations in the function of any one of the protein kinases could leadto cell immortality or premature cell death.

The term “administering” relates to a method of incorporating a compoundinto cells or tissues of an organism. The abnormal condition can beprevented or treated when the cells or tissues of the organism existwithin the organism or outside of the organism. Cells existing outsidethe organism can be maintained or grown in cell culture dishes. Forcells harbored within the organism, many techniques exist in the art toadminister compounds, including (but not limited to) oral, parenteral,dermal, injection, and aerosol applications. For cells outside of theorganism, multiple techniques exist in the art to administer thecompounds, including (but not limited to) cell microinjectiontechniques, transformation techniques and carrier techniques.

The abnormal condition can also be prevented or treated by administeringa compound to a group of cells having an aberration in a signaltransduction pathway to an organism. The effect of administering acompound on organism function can then be monitored. The organism ispreferably a mouse, rat, rabbit, guinea pig or goat, more preferably amonkey or ape, and most preferably a human.

By “amplification” it is meant increased numbers of DNA or RNA in a cellcompared with normal cells. “Amplification” as it refers to RNA can bethe detectable presence of RNA in cells, since in some normal cellsthere is no basal expression of RNA. In other normal cells, a basallevel of expression exists, therefore in these cases amplification isthe detection of at least 1-2-fold, and preferably more, compared to thebasal level.

The amino acid sequences are presented in the amino to carboxydirection, from left to right. The amino and carboxy groups are notpresented in the sequence. The nucleotide sequences are presented bysingle strand only, in the 5′ to 3′ direction, from left to right.Nucleotides and amino acids are represented in the manner recommended bythe IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids)by three letters code.

Nucleic Acids

Genomic DNA of the invention comprises the protein-coding region for apolypeptide of the invention and is also intended to include allelicvariants thereof. It is widely understood that, for many genes, genomicDNA is transcribed into RNA transcripts that undergo one or moresplicing events wherein intron (i.e., non-coding regions) of thetranscripts are removed, or “spliced out.” RNA transcripts that can bespliced by alternative mechanisms, and therefore be subject to removalof different RNA sequences but still encode a NgR polypeptide, arereferred to in the art as splice variants which are embraced by theinvention. Splice variants comprehended by the invention therefore areencoded by the same original genomic DNA sequences but arise fromdistinct mRNA transcripts. Allelic variants are modified forms of awild-type gene sequence, the modification resulting from recombinationduring chromosomal segregation or exposure to conditions which give riseto genetic mutation. Allelic variants, like wild-type genes, arenaturally occurring sequences (as opposed to non-naturally occurringvariants arising from in vitro manipulation).

The invention also comprehends cDNA that is obtained through reversetranscription of an RNA polynucleotide encoding NgR (conventionallyfollowed by second-strand synthesis of a complementary strand to providea double-stranded DNA).

Preferred DNA sequences encoding a human NgR polypeptide is set out inSEQ ID NOs:1 and 13. A preferred DNA of the invention comprises a doublestranded molecule comprising the coding molecule (i.e., the “codingstrand”) along with the complementary molecule (the “non-coding strand”or “complement”) having a sequence unambiguously deducible from thecoding strand according to Watson-Crick base-pairing rules for DNA. Alsopreferred are other polynucleotides encoding NgR polypeptides, as shownin SEQ ID NO:3, which comprises murine NgR homolog, NgR3.

Also preferred are nucleotide sequences that encode at least a portionof a NgR polypeptide that has at least one biological function of a NgR.More preferred are nucleotide sequences that encode a portion of NgRthat encodes at least the mature NgR without the hydrophobic C-terminalGPI signal. Also preferred are nucleotide sequences that encode theportion of NgR that encodes at least the ligand-binding region of NgR.

The invention further embraces other species, preferably mammalian,homologs of the human NgR DNA. Species homologs, sometimes referred toas “orthologs,” in general, share at least 35%, at least 40%, at least45%, at least 50%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% homology with human DNA of the invention.Generally, percent sequence “homology” with respect to polynucleotidesof the invention may be calculated as the percentage of nucleotide basesin the candidate sequence that are identical to nucleotides in the NgRsequences set forth in SEQ ID NOs:1, 3 or 13, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

The polynucleotide sequence information provided by the invention makespossible large-scale expression of the encoded polypeptide by techniqueswell known and routinely practiced in the art. Polynucleotides of theinvention also permit identification and isolation of polynucleotidesencoding related NgR polypeptides, such as human allelic variants andspecies homologs, by well-known techniques including Southern and/orNorthern hybridization, and polymerase chain reaction (PCR). Examples ofrelated polynucleotides include human and non-human genomic sequences,including allelic variants, as well as polynucleotides encodingpolypeptides homologous to NgR and structurally related polypeptidessharing one or more biological, immunological, and/or physicalproperties of NgR. Non-human species genes encoding proteins homologousto NgR can also be identified by Southern and/or PCR analysis and areuseful in animal models for NgR disorders. Knowledge of the sequence ofa human NgR DNA also makes possible through use of Southernhybridization or polymerase chain reaction (PCR) the identification ofgenomic DNA sequences encoding NgR expression control regulatorysequences such as promoters, operators, enhancers, repressors, and thelike. Polynucleotides of the invention are also useful in hybridizationassays to detect the capacity of cells to express NgR Polynucleotides ofthe invention may also provide a basis for diagnostic methods useful foridentifying a genetic alteration(s) in a NgR locus that underlies adisease state or states, which information is useful both for diagnosisand for selection of therapeutic strategies.

The disclosure herein of a full-length polynucleotide encoding a NgRpolypeptide makes readily available to the worker of ordinary skill inthe art every possible fragment of the full-length polynucleotide. Theinvention, therefore, provides fragments of NgR-encoding polynucleotidescomprising at least 6, and preferably at least 14, 16, 18, 20, 25, 50,or 75 consecutive nucleotides of a polynucleotide encoding NgR.Preferably, fragments of polynucleotides of the invention comprisesequences unique to the NgR-encoding polynucleotide sequence, andtherefore hybridize under highly stringent or moderately stringentconditions only (i.e., “specifically”) to polynucleotides encoding NgR(or fragments thereof). Polynucleotide fragments of genomic sequences ofthe invention comprise not only sequences unique to the coding region,but also include fragments of the full-length sequence derived fromintrons, regulatory regions, and/or other non-translated sequences.Sequences unique to polynucleotides of the invention are recognizablethrough sequence comparison to other known polynucleotides, and can beidentified through use of alignment programs routinely utilized in theart, e.g., those made available in public sequence databases. Suchsequences also are recognizable from Southern hybridization analyses todetermine the number of fragments of genomic DNA to which apolynucleotide will hybridize. Polynucleotides of the invention can belabeled in a manner that permits their detection, including radioactive,fluorescent and enzymatic labeling.

Fragments of polynucleotides are particularly useful as probes fordetection of full-length or fragment of NgR polynucleotides. One or morepolynucleotides can be included in kits that are used to detect thepresence of a polynucleotide encoding NgR, or used to detect variationsin a polynucleotide sequence encoding NgR.

The invention also embraces DNAs encoding NgR polypeptides thathybridize under moderately stringent or high stringency conditions tothe noncoding strand, or complement, of the polynucleotide in any of SEQID NOs:1 or 3

Stringent conditions are known to those skilled in the art and can befound in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.(1989), 6.3.1?6.3.6. Preferably, the conditions are such that sequencesat least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homologous toeach other typically remain hybridized to each other. A non-limitingexample of stringent hybridization conditions is hybridization in a highsalt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%PVP, 0.02% Ficoll, 0.02% BSA and 500 mg/ml denatured salmon sperm DNA at65° C. This hybridization is followed by one or more washes in 0.2×SSC,0.01% BSA at 50° C. An isolated nucleic acid molecule of the inventionthat hybridizes under stringent conditions to the sequence of SEQ IDNOs:1 or 3 corresponds to a naturally occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein). As used herein, “stringenthybridization conditions” means: 42° C. in a hybridization solutioncomprising 50% formamide, 1% SDS, 1 M NaCl, 10% (wt/vol) dextransulfate, and washing twice for 30 minutes at 60° C. in a wash solutioncomprising 0.1×SSC and 1% SDS.

Vectors

Another aspect of the present invention is directed to vectors, orrecombinant expression vectors, comprising any of the nucleic acidmolecules described above. Vectors are used herein either to amplify DNAor RNA encoding NgR and/or to express DNA which encodes NgR. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), that serveequivalent functions.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein, (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67, 3140), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione-S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69, 301-315) and pET 11d(Studier et al., GENE EXRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS ENZYMOLOGY 185, Acadermic Press, SanDiego, Calif. (1990) 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20, 2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the NgR expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J. 6, 229-234), pMFa(Kurjan and Herskowitz (1982) Cell 30, 933-943), pJRY88 (Schultz et al.,(1987) Gene 54, 113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, NgR can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith et al., (1983) Mol. Cell. Biol. 3, 2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170, 31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329, 840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6, 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., (Eds.) MOLECULAR CLONING: A LABORATORY MANUAL. 2^(nd)Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1, 268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43, 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8, 729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33, 729-740; Queen andBaltimore (1983) Cell 33, 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86, 5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230, 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264, 166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss (1990)Science 249, 374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3, 537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense NgR mRNA. Regulatory sequences operatively linked to anucleic acid cloned in the antisense orientation can be chosen thatdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen that direct constitutive,tissue-specific or cell-type-specific expression of antisense RNA Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al., Antisense RNA asa molecular tool for genetic analysis, REVIEWS-TRENDS IN GENETICS, Vol.1(1) 1986.

Preferred vectors include, but are not limited to, plasmids, phages,cosmids, episomes, viral particles or viruses and integratable DNAfragments (i.e., fragments integratable into the host genome byhomologous recombination). Preferred viral particles include, but arenot limited to, adenoviruses, baculoviruses, parvoviruses,herpesviruses, poxviruses, adeno-associated viruses, Semliki Forestviruses, vaccinia viruses and retroviruses. Preferred expression vectorsinclude, but are not limited to, pcDNA3 (Invitrogen) and pSVL (PharmaciaBiotech). Other expression vectors include, but are not limited to,pSPORT™ vectors, pGEM™ vectors (Promega), pPROEXvectors™ (LTI, Bethesda,Md.), Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™(Invitrogen) and pYES2™ (Invitrogen).

Preferred expression vectors are replicable DNA constructs in which aDNA sequence encoding NgR is operably linked or connected to suitablecontrol sequences capable of effecting the expression of the NgR in asuitable host. DNA regions are operably linked or connected when theyare functionally related to each other. For example, a promoter isoperably linked or connected to a coding sequence if it controls thetranscription of the sequence. Amplification vectors do not requireexpression control domains, but rather need only the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants. The needfor control sequences in the expression vector will vary depending uponthe host selected and the transformation method chosen. Generally,control sequences include, but are not limited to a transcriptionalpromoter, enhancers, an optional operator sequence to controltranscription, polyadenylation signals, a sequence encoding suitablemRNA ribosomal binding and sequences which control the termination oftranscription and translation. Such regulatory sequences are described,for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., NgR proteins, mutant forms ofNgR, fusion proteins, etc.).

Preferred vectors preferably contain a promoter that is recognized bythe host organism. The promoter sequences of the present invention maybe prokaryotic, eukaryotic or viral. Examples of suitable prokaryoticsequences include the PR and PL promoters of bacteriophage lambda (THEBACTERIOPHAGE LAMBDA, Hershey, A. D. (Ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1973), which is incorporatedherein by reference in its entirety; LAMBDA II, Hendrix, R. W. (Ed.),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1980),which is incorporated herein by reference in its entirety); the trp,recA, heat shock, and lacZ promoters of E. coli and the SV40 earlypromoter (Benoist et al., (1981) Nature 290, 304-310, which isincorporated herein by reference in its entirety). Additional promotersinclude, but are not limited to, mouse mammary tumor virus, longterminal repeat of human immunodeficiency virus, maloney virus,cytomegalovirus immediate early promoter, Epstein Barr virus, Roussarcoma virus, human actin, human myosin, human hemoglobin, human musclecreatine and human metallothionein.

Additional regulatory sequences can also be included in preferredvectors. Preferred examples of suitable regulatory sequences arerepresented by the Shine-Dalgarno sequence of the replicase gene of thephage MS-2 and of the gene cII of bacteriophage lambda. TheShine-Dalgarno sequence may be directly followed by DNA encoding NgR andresult in the expression of the mature NgR protein.

Moreover, suitable expression vectors can include an appropriate markerthat allows the screening of the transformed host cells. Thetransformation of the selected host is carried out using any one of thevarious techniques well known to the expert in the art and described inSambrook et al., supra.

An origin of replication can also be provided either by construction ofthe vector to include an exogenous origin or may be provided by the hostcell chromosomal replication mechanism. If the vector is integrated intothe host cell chromosome, the latter may be sufficient. Alternatively,rather than using vectors which contain viral origins of replication,one skilled in the art can transform mammalian cells by the method ofco-transformation with a selectable marker and NgR DNA. An example of asuitable marker is dihydrofolate reductase (DHFR) or thymidine kinase(see, U.S. Pat. No. 4,399,216).

Nucleotide sequences encoding NgR may be recombined with vector DNA inaccordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining and ligationwith appropriate ligases. Techniques for such manipulation are disclosedby Sambrook et al., supra and are well known in the art. Methods forconstruction of mammalian expression vectors are disclosed in, forexample, Okayama et al., (1983) Mol. Cell. Biol. 3:280, Cosman et al.(1986) Mol. Immunol. 23:935, Cosman et al., (1984) Nature 312:768,EP-A-0367566, and WO 91/18982, each of which is incorporated herein byreference in its entirety.

Host Cells and Transformed Host Cells

According to another aspect of the invention, host cells are provided,including prokaryotic and eukaryotic cells, comprising a polynucleotideof the invention (or vector of the invention) in a manner that permitsexpression of the encoded NgR polypeptide. Preferably, the cell produceslittle or no endogenous NgR polypeptide. Polynucleotides of theinvention may be introduced into the host cell as part of a circularplasmid, or as linear DNA comprising an isolated protein coding regionor a viral vector. Methods for introducing DNA into the host cell thatare well known and routinely practiced in the art includetransformation, transfection, electroporation, nuclear injection, orfusion with carriers such as liposomes, micelles, ghost cells andprotoplasts. Expression systems of the invention include bacterial,yeast, fungal, plant, insect, invertebrate, vertebrate and mammaliancells systems.

Host cells of the invention are a valuable source of immunogen fordevelopment of antibodies specifically immunoreactive with NgR. Hostcells of the invention are also useful in methods for the large-scaleproduction of NgR polypeptides wherein the cells are grown in a suitableculture medium and the desired polypeptide products are isolated fromthe cells, or from the medium in which the cells are grown, bypurification methods known in the art, e.g., conventionalchromatographic methods including immunoaffinity chromatography,receptor affinity chromatography, hydrophobic interactionchromatography, lectin affinity chromatography, size exclusionfiltration, cation or anion exchange chromatography, high pressureliquid chromatography (HPLC), reverse phase HPLC, and the like. Stillother methods of purification include those methods wherein the desiredprotein is expressed and purified as a fusion protein having a specifictag, label or chelating moiety that is recognized by a specific bindingpartner or agent. The purified protein can be cleaved to yield thedesired protein, or can be left as an intact fusion protein. Cleavage ofthe fusion component may produce a form of the desired protein havingadditional amino acid residues as a result of the cleavage process.

Knowledge of NgR DNA sequences allows for modification of cells topermit, or increase, expression of endogenous NgR Cells can be modified(e.g., by homologous recombination) to provide increased expression byreplacing, in whole or in part, the naturally occurring NgR promoterwith all or part of a heterologous promoter so that the cells expressNgR at higher levels. The heterologous promoter is inserted in such amanner that it is operatively linked to endogenous NgR encodingsequences. (See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO 92120808, and PCTInternational Publication No. WO 91/09955.) It is also contemplatedthat, in addition to heterologous promoter DNA, amplifiable marker DNA(e.g., ada, dhfr, and the multifunctional CAD gene which encodescarbamoyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA If linked to the NgR coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the NgR coding sequences in the cells.

The DNA sequence information provided by the present invention alsomakes possible the development (e.g., by homologous recombination or“knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989)) ofanimals that fail to express functional NgR or that express a variant ofNgR. Such animals (especially small laboratory animals such as rats,rabbits and mice) are useful as models for studying the in vivoactivities of NgR and modulators of NgR.

Suitable host cells for expression of the polypeptides of the inventioninclude, but are not limited to, prokaryotes, yeast, and eukaryotes. Ifa prokaryotic expression vector is employed, then the appropriate hostcell would be any prokaryotic cell capable of expressing the clonedsequences. Suitable prokaryotic cells include, but are not limited to,bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas,Streptomyces and Staphylococcus.

If a eukaryotic expression vector is employed, then the appropriate hostcell would be any eukaryotic cell capable of expressing the clonedsequence. Preferably, eukaryotic cells are cells of higher eukaryotes.Suitable eukaryotic cells include, but are not limited to, non-humanmammalian tissue culture cells and human tissue culture cells. Preferredhost cells include, but are not limited to, insect cells, HeLa cells,Chinese hamster ovary cells (CHO cells), African green monkey kidneycells (COS cells), human 293 cells, and murine 3T3 fibroblasts.Propagation of such cells in cell culture has become a routine procedure(see, Tissue Culture, Academic Press, Kruse and Patterson, Eds. (1973),which is incorporated herein by reference in its entirety).

In addition, a yeast cell may be employed as a host cell. Preferredyeast cells include, but are not limited to, the genera Saccharomyces,Pichia and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P.pastoris. Preferred yeast vectors can contain an origin of replicationsequence from a 2T yeast plasmid, an autonomously replication sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination and a selectable marker gene Shuttle vectorsfor replication in both yeast and E. coli are also included herein.

Alternatively, insect cells may be used as host cells. In a preferredembodiment, the polypeptides of the invention are expressed using abaculovirus expression system (see, Luckow et al., Bio/Technology, 1988,6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, O'Rielly etal. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No.4,879,236, each of which is incorporated herein by reference in itsentirety). In addition, the MAXBAC™ complete baculovirus expressionsystem (Invitrogen) can, for example, be used for production in insectcells.

Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin, dihydrofolate reductase (DHFR) and methotrexate.Nucleic acid encoding a selectable marker can be introduced into a hostcell on the same vector as that encoding NgR or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

In a preferred embodiment, the polypeptides of the invention, includingforms of NgR2 and NgR3, soluble forms of NgR, chimeric NgR polypeptides,NgR/Ig fusions and fragments and variations of each of the above areexpressed in Chinese Hamster Ovary (CHO) cells.

In order to introduce the DNA fragment coding for the NgR protein orpolypeptide into the CHO cell to express the recombinant NgR protein orpolypeptide, it is necessary to construct the expression vector.

The vectors for CHO expression include, but are not limited to, pA1-11,pXT1, pRc/CMV, pRc/RSV and pcDNAINeo. The promoter is not specificallylimited provided it effectively promotes expression in CHO cells.Examples of suitable promoters are: SRα, SV40, LTR, CMV, and HSV-TK. Ofthese, CMV and Srα promoters are preferred.

In addition to the above-mentioned promoters, the expression vectors maycontain enhancers, splicing signals, polyadenylation signals, selectablemarkers and an SV40 replication origin. Suitable selectable markersinclude, but are not limited to the dihydrofolate reductase (DHFR) genewhich provides resistance to methotrexate (MTX), the ampicillinresistance gene, and the neomycin resistance gene.

Examples of the expression vectors each containing the DNA coding forNgR, portions, fragments and soluble constructs thereof, include thevector (such as one described above), into which the promoter isoperably linked (preferably upstream) to the nucleotide sequenceencoding the desired NgR construct; a polyadenylation signal downstreamfrom the nucleotide sequence encoding the NgR construct; and,preferably, the vector includes an operable DHFR gene. Preferably, theampicillin resistant gene is also operably contained in the vector.

CHO cell lacking the DHFR gene (Urlaub, G. et al., (1980) Proc. Natl.Acad. Sci. USA 77, 4216-4220) and CHO-K1 (Proc. Natl. Acad. Sci. USA 60,1275 (1968)) are suitable for use.

The NgR expression vectors prepared as above are introduced into CHOcells by any known method, including, but not limited to the calciumphosphate method (Graham and van der Eb (1973) Virol. 52, 456-467) andelectroporation (Nuemann et al., (1982) EMBO J. 1, 841-845).

Transformants carrying the expression vectors are selected based on theabove-mentioned selectable markers. Repeated clonal selection of thetransformants using the selectable markers allows selection of stablecell lines having high expression of the NgR constructs. Increased MTXconcentrations in the selection medium allows gene amplification andgreater expression of the desired protein. The CHO cell containing therecombinant NgR can be produced by cultivating the CHO cells containingthe NR expression vectors constitutively expressing the NgR constructs.

Media used in cultivating CHO cells includes DMEM medium supplementedwith about 0.5 to 20% fetal calf serum, DMEM medium and RPMI1640 medium.The pH of the medium is preferably about 6 to 8. Cultivation ispreferably at about 30 to 40° C. for about 15 to 72 hours with aeration.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) NgR protein.Accordingly, the invention further provides methods for producing NgRprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding NgR has been introduced) in asuitable medium such that NgR protein is produced. In anotherembodiment, the method further comprises isolating NgR from the mediumor the host cell.

In situations where the NgR polypeptide will be found primarilyintracellularly, intracellular material (including inclusion bodies forGram-negative bacteria) can be extracted from the host cell using anystandard technique known to one of ordinary skill in the art. Suchmethods would encompass, by way of example and not by way of limitation,lysing the host cells to release the contents of the periplasm/cytoplasmby French press, homogenization and/or sonication followed bycentrifugation.

If the NgR polypeptide has formed inclusion bodies in the cytosol, suchinclusion bodies may frequently bind to the inner and/or outer cellularmembranes. Upon centrifugation, the inclusion bodies will be foundprimarily in the pellet material. The pellet material can then betreated at pH extremes or with one or more chaotropic agents such as adetergent, guanidine, guanidine derivatives, urea, or urea derivativesin the presence of a reducing agent such as dithiothreitol at alkalinepH or tris-carboxyethyl phosphine at acid pH to release, break apart andsolubilize the inclusion bodies. Once solubilized, NgR polypeptide canbe analyzed using gel electrophoresis, immunoprecipitation or the like.Various methods of isolating the NgR polypeptide would be apparent toone of ordinary skill in the art, for example, isolation may beaccomplished using standard methods such as those set forth below and inMarston et al (1990) Meth. Enzymol. 182, 264-275 (incorporated byreference herein in its entirety).

If isolated NgR polypeptide is not biologically active following theisolation procedure employed, various methods for “refolding” orconverting the polypeptide to its tertiary structure and generatingdisulfide linkages, can be used to restore biological activity. Methodsknown to one of ordinary skill in the art include adjusting the pH ofthe solubilized polypeptide to a pH usually above 7 and in the presenceof a particular concentration of a chaotrope. The selection of chaotropeis very similar to the choices used for inclusion body solubilizationbut usually at a lower concentration and is not necessarily the samechaotrope as used for the solubilization. It may be required to employ areducing agent or the reducing agent plus its oxidized form in aspecific ratio, to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridge(s). Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol (DTT)/dithiane DTT, 2-mercaptoethanol (bME)/dithio-b(ME).To increase the efficiency of the refolding, it may be necessary toemploy a cosolvent, such as glycerol, polyethylene glycol of variousmolecular weights and arginine.

Transgenic Animals

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichNgR-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous NgRsequences have been introduced into their genome or homologousrecombinant animals in which endogenous NgR sequences have been altered.Such animals are useful for studying the function and/or activity of NgRand for identifying and/or evaluating modulators of NgR activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, etc. A transgene is exogenous DNA that isintegrated into the genome of a cell from which a transgenic animaldevelops and that remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous NgR gene has been altered byhomologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingNgR-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanNgR DNA sequence of SEQ ID NOs:1 or 3 can be introduced as a transgeneinto the genome of a non-human animal. Alternatively, a nonhuman homologof the human NgR gene, such as a mouse NgR gene, can be isolated basedon hybridization to the human NgR cDNA (described further above) andused as a transgene. Intronic sequences and polyadenylation signals canalso be included in the transgene to increase the efficiency ofexpression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the NgR transgene to direct expression of NgRprotein to particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; andHogan 1986, in MANIPULATING THE MOUSE EMBRYO, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Similar methods are used forproduction of other transgenic animals. A transgenic founder animal canbe identified based upon the presence of the NgR transgene in its genomeand/or expression of NgR mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding NgR can further be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a NgR gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the NgR gene. The NgR gene can be a human gene(e.g., SEQ ID NOs:1 or 13), but more preferably, is a non-human homologof a human NgR gene. For example, a mouse homolog of human NgR gene ofSEQ ID NOs:1 or 13 can be used to construct a homologous recombinationvector suitable for altering an endogenous NgR gene in the mouse genome.In one embodiment, the vector is designed such that, upon homologousrecombination, the endogenous NgR gene is functionally disrupted (i.e.,no longer encodes a functional protein; also referred to as a “knockout” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous NgR gene is mutated or otherwise alteredbut still encodes functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenousNgR protein). In the homologous recombination vector, the alteredportion of the NgR gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the NgR gene to allow for homologous recombination tooccur between the exogenous NgR gene carried by the vector and anendogenous NgR gene in an embryonic stem cell. The additional flankingNgR nucleic acid is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector.See e.g., Thomas et al. (1987) Cell 51:503 for a description ofhomologous recombination vectors. The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced NgR gene has homologously recombined with the endogenousNgR gene are selected (see e.g., Li et al. (1992) Cell 69:915).

The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. See e.g., Bradley 1987,In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A Practical Approach,Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley (1991) Curr. Opin. Biotechnol. 2:823-829; PCTInternational Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968;and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedthat contain selected systems that allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. SciUSA 89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355. If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Antisense

Also provided by the invention are antisense polynucleotides thatrecognize and hybridize to NgR polynucleotides. Full-length and fragmentantisense polynucleotides are provided. Fragment antisense molecules ofthe invention include (i) those that specifically recognize andhybridize to NgR RNA (as determined by sequence comparison of DNAencoding NgR to DNA encoding other known molecules). Identification ofsequences unique to NgR encoding polynucleotides can be deduced throughuse of any publicly available sequence database, and/or through use ofcommercially available sequence comparison programs. Afteridentification of the desired sequences, isolation through restrictiondigestion or amplification using any of the various polymerase chainreaction techniques well known in the art can be performed. Antisensepolynucleotides are particularly relevant to regulating expression ofNgR by those cells expressing NgR mRNA.

Antisense oligonucleotides, or fragments of a nucleotide sequence setforth in SEQ ID NO:1, 3, 13 or sequences complementary or homologousthereto, derived from the nucleotide sequences of the present inventionencoding NgR are useful as diagnostic tools for probing gene expressionin various tissues. For example, tissue can be probed in situ witholigonucleotide probes carrying detectable groups by conventionalautoradiography techniques to investigate native expression of thisenzyme or pathological conditions relating thereto. In specific aspects,antisense nucleic acid molecules are provided that comprise a sequencecomplementary to at least about 10, 25, 50, 100, 250 or 500 nucleotidesor an entire NgR coding strand, or to only a portion thereof. Nucleicacid molecules encoding fragments, homologs, derivatives and analogs ofa NgR protein of SEQ ID NO:2, 4 or 14 or antisense nucleic acidscomplementary to a NgR nucleic acid sequence of SEQ ID NOs:1, 3 or 13are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingNgR. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., the protein coding region of human NgR corresponds to the codingregion SEQ ID NO:1, 3 or 13). In another embodiment, the antisensenucleic acid molecule is antisense to a “noncoding region” of the codingstrand of a nucleotide sequence encoding NgR. The term “noncodingregion” refers to 5′ and 3′ sequences which flank the coding region thatare not translated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Antisense oligonucleotides are preferably directed to regulatory regionsof a nucleotide sequence of SEQ ID NO:1, 3, 13 or mRNA correspondingthereto, including, but not limited to, the initiation codon, TATA box,enhancer sequences, and the like. Given the coding strand sequencesencoding NgR disclosed herein (e.g., SEQ ID NO:1, 3 or 13), antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick or Hoogsteen base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of NgR mRNA,but more preferably is an oligonucleotide that is antisense to only aportion of the coding or noncoding region of NgR mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of NgR mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis or enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,S-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention (preferablyoligonucleotides of 10 to 20 nucleotides in length) are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a NgR proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. Suppression of NgR expression ateither the transcriptional or translational level is useful to generatecellular or animal models for diseases/conditions characterized byaberrant NgR expression. The hybridization can be by conventionalnucleotide complementarity to form a stable duplex, or, for example, inthe case of an antisense nucleic acid molecule that binds to DNAduplexes, through specific interactions in the major groove of thedouble helix.

Phosphorothioate and methylphosphonate antisense oligonucleotides arespecifically contemplated for therapeutic use by the invention. Theantisense oligonucleotides may be further modified by addingpoly-L-lysine, transferrin polylysine or cholesterol moieties at their5′ end.

An example of a route of administration of antisense nucleic acidmolecules of the invention includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies that bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of antisense molecules, vector constructsin which the antisense nucleic acid molecule is placed under the controlof a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., (1987) Nucleic Acids Res. 15, 6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15,6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBSLett. 215, 327-330).

The NgR sequences taught in the present invention facilitate the designof novel transcription factors for modulating NgR expression in nativecells and animals, and cells transformed or transfected with NgRpolynucleotides. For example, the Cys₂-His₂ zinc finger proteins, whichbind DNA via their zinc finger domains, have been shown to be amenableto structural changes that lead to the recognition of different targetsequences. These artificial zinc finger proteins recognize specifictarget sites with high affinity and low dissociation constants, and areable to act as gene switches to modulate gene expression. Knowledge ofthe particular NgR target sequence of the present invention facilitatesthe engineering of zinc finger proteins specific for the target sequenceusing known methods such as a combination of structure-based modelingand screening of phage display libraries (Segal et al., (1999) Proc.Natl. Acad. Sci. USA 96, 2758-2763; Liu et al., (1997) Proc. Nail. Acad.Sci. USA 94, 5525-5530; Greisman et al. (1997) Science 275, 657-661;Choo et al., (1997) J. Mol. Biol. 273, 525-532). Each zinc finger domainusually recognizes three or more base pairs. Since a recognitionsequence of 18 base pairs is generally sufficient in length to render itunique in any known genome, a zinc finger protein consisting of 6 tandemrepeats of zinc fingers would be expected to ensure specificity for aparticular sequence (Segal et al., (1999), above). The artificial zincfinger repeats, designed based on the promoter of NgR sequences, arefused to activation or repression domains to promote or suppress NgRexpression (Liu et al., (1997), above). The promoter of NgR may beobtained by standard methods known to one of ordinary skill in the artwith the disclosure contained herein and knowledge of the NgR sequence.Alternatively, the zinc finger domains can be fused to the TATAbox-binding factor (TBP) with varying lengths of linker region betweenthe zinc finger peptide and the TBP to create either transcriptionalactivators or repressors (Kim et al., (1997) Proc. Natl. Acad. Sci. USA94, 3616-3620. Such proteins and polynucleotides that encode them, haveutility for modulating NgR expression in vivo in both native cells,animals and humans; and/or cells transfected with NgR-encodingsequences. The novel transcription factor can be delivered to the targetcells by transfecting constructs that express the transcription factor(gene therapy), or by introducing the protein. Engineered zinc fingerproteins can also be designed to bind RNA sequences for use intherapeutics as alternatives to antisense or catalytic RNA methods(McColl et al., (1997) Proc. Natl. Acad. Sci. USA 96, 9521-9526); Wu etal., (1995) Proc. Natl. Acad. Sci. USA 92, 344-348). The presentinvention contemplates methods of designing such transcription factorsbased on the gene sequence of the invention, as well as customized zincfinger proteins, that are useful to modulate NgR expression in cells(native or transformed) whose genetic complement includes thesesequences.

Ribozymes and PNA Moieties

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes, described in Haselhoff andGerlach (1988) Nature 334, 585-591) can be used to catalytically cleaveNgR mRNA transcripts to thereby inhibit translation of NgR RNA. Aribozyme having specificity for a NgR-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a NgR DNA disclosedherein (i.e., SEQ ID NOs:1, 3 or 13). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a NgR-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,Ng mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel etal., (1993) Science 261, 1411-1418.

Alternatively, NgR gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the NgR(e.g., the NgR promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the NgR gene in target cells.See generally, Helene (1991) Anticancer Drug Des. 6: 569-584; Helene. etal., (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) BioEssays14, 807-815.

In various embodiments, the nucleic acids of NgR can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup et al., (1996) Bioorg. Med.Chem. Lett. 4, 5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al., (1996) above; Perry-O'Keefe etal., (1996) Proc. Nail. Acad. Sci. USA 93, 14670-14675.

PNAs of NgR can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofNgR can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases (Hyrup (1996), above); or as probes or primers for DNAsequence and hybridization (Hyrup et al., (1996), above; Perry-O'Keefe(1996), above).

In another embodiment, PNAs of NgR can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of NgR can be generated that may combinethe advantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNase H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996), above).The synthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), above and Finn et al. (1996) Nucleic Acids Res. 24, 3357-3363.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl) amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al. (1989) Nucleic Acids Res. 17, 973-988). PNA monomers are thencoupled in a stepwise manner to produce a chimeric molecule with a 5′PNA segment and a 3′ DNA segment (Finn et al. (1996), above).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg. Med.Chem. Lett. 5:1119-1124.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (seeLetsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86, 6553-6556;Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84, 648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization triggered cleavage agents (see, e.g., Krolet al., (1988) Biotechniques 6, 958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5, 539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, etc.

Automated sequencing methods can be used to obtain or verify thenucleotide sequence of NgR. The NgR nucleotide sequences of the presentinvention are believed to be 100% accurate. However, as is known in theart, nucleotide sequence obtained by automated methods may contain someerrors. Nucleotide sequences determined by automation are typically atleast about 90%, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of a given nucleicacid molecule. The actual sequence may be more precisely determinedusing manual sequencing methods, which are well known in the art. Anerror in a sequence which results in an insertion or deletion of one ormore nucleotides may result in a frame shift in translation such thatthe predicted amino acid sequence will differ from that which would bepredicted from the actual nucleotide sequence of the nucleic acidmolecule, starting at the point of the mutation.

Polypeptides

The invention also provides purified and isolated mammalian NgRpolypeptides encoded by a polynucleotide of the invention. Presentlypreferred is a human NgR polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:2 or SEQ ID NO:14. Another preferred embodimentis a mouse NgR polypeptide comprising the amino acid sequence of NgR3,as set forth in SEQ ID NO:4.

One aspect of the invention pertains to isolated NgR proteins, andbiologically active portions thereof, or derivatives, fragments, analogsor homologs thereof. Also provided are polypeptide fragments suitablefor use as immunogens to raise anti-NgR antibodies. Preferably,fragments of NgR proteins comprise at least one biological activity ofNgR. In one embodiment, native NgR proteins can be isolated from cellsor tissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, NgR proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a NgR protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

The invention also embraces polypeptides that have at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, at least 50% or atleast 45% identity and/or homology to the preferred polypeptide of theinvention. In addition, the invention embraces polypeptides having theconsensus sequence shown in SEQ ID NO:6, shown in Table 5) excluding thepreviously characterized NgR (“NgR1”), and polypeptides comprising atleast about 90% of the consensus sequence.

The term “percentage of sequence identity” is calculated by comparingtwo optimally aligned sequences over that region of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in theregion of comparison (i.e., the window size), and multiplying the resultby 100 to yield the percentage of sequence identity. The term“substantial identity” as used herein denotes a characteristic of apolynucleotide sequence, wherein the polynucleotide comprises a sequencethat has at least 80 percent sequence identity, preferably at least 85percent identity and often 90 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison region.

In one aspect, percent homology is calculated as the percentage of aminoacid residues in the smaller of two sequences which align with identicalamino acid residue in the sequence being compared, when four gaps in alength of 100 amino acids may be introduced to maximize alignment(Dayhoff, in ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, Vol. 5, p. 124,National Biochemical Research Foundation, Washington, D.C. (1972),incorporated herein by reference).

A determination of homology or identity is typically made by a computerhomology program known in the art. An exemplary program is the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for UNIXGenetics Computer Group, University Research Park, Madison, Wis.) usingthe default settings, which uses the algorithm of Smith and Waterman(Adv. Appl. Math, 1981, 2, 482-489, which in incorporated herein byreference in its entirety). Employing the GAP software provided in theGCG program package, (see Needleman and Wunsch (1970) J. Mol. Biol. 48,443-453) the following settings for nucleic acid sequence comparison maybe used: GAP creation penalty of 5.0 and GAP extension penalty of 0.3,the coding region of the analogous nucleic acid sequences referred toabove exhibits a degree of identity preferably of at least 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNAsequence shown in SEQ ID NOs:1, 3 or 13. BestFit was originally writtenfor Version 1.0 by Paul Haeberli from a careful reading of the papers byNeedleman and Wunsch (1970), above, and Smith and Waterman (1981),above. The following Bestfit settings for nucleic acid sequencecomparison may be used: GAP creation penalty of 8.0 and GAP extensionpenalty of 2, the coding region of the analogous nucleic acid sequencesreferred to above exhibits a degree of identity preferably of at least70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, with the CDS (encoding) partof the amino acid sequence shown in SEQ ID NOs:2, 4 or 14.

Alternatively, homology may be determined by hybridization analysiswherein a nucleic acid sequence is hybridized to the complement of asequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. See e.g. Ausubel, etal., Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,New York, N.Y., 1993, and below.

Polypeptides of the invention may be isolated from natural cell sourcesor may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the NgRprotein is derived, or substantially free from chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of NgR protein in whichthe protein is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof NgR protein having less than about 30% (by dry weight) of non-NgRprotein (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-NgR protein, still more preferablyless than about 10% of non-NgR protein, and most preferably less thanabout 5% non-NgR protein. When the NgR protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of NgR protein in which the protein isseparated from chemical precursors or other chemicals that are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of NgR protein having less than about 30% (by dry weight)of chemical precursors or non-NgR chemicals, more preferably less thanabout 20% chemical precursors or non-NgR chemicals, still morepreferably less than about 10% chemical precursors or non-NgR chemicals,and most preferably less than about 5% chemical precursors or non-NgRchemicals.

Biologically active portions of a NgR protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the NgR protein, e.g., the amino acidsequence shown in SEQ ID NO:2, 4 or 14 that include fewer amino acidsthan the full length NgR proteins, and exhibit at least one activity ofa NgR protein. Typically, biologically active portions comprise a domainor motif with at least one activity of the NgR protein. A biologicallyactive portion of a NgR protein can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length.

A biologically active portion of a NgR protein of the present inventionmay contain at least one of the features that is conserved between theNgR proteins (e.g., a conserved cysteine as the N-terminus of the matureprotein, four conserved cysteines in the N-terminus before aleucine-rich region, four conserved cysteines C-terminal with respect toa leucine repeat region, eight leucine-rich repeats, and a hydrophobicC-terminus). An alternative biologically active portion of a NgR proteinmay contain at least two of the above-identified domains. Anotherbiologically active portion of a NgR protein may contain at least threeof the above-identified domains. Yet another biologically active portionof a NgR protein of the present invention may contain at least four ofthe above-identified domains.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native NgRprotein.

In an embodiment, the NgR protein has an amino acid sequence shown inSEQ ID NO:2, 4 or 14. In other embodiments, the NgR protein issubstantially homologous to SEQ ID NO:2, 4 or 14 and retains thefunctional activity of the protein of SEQ ID NO:2, 4 or 14, yet differsin amino acid sequence due to natural allelic variation or mutagenesis,as described in detail below.

Accordingly, in another embodiment, the NgR protein is a protein thatcomprises an amino acid sequence at least about 45% homologous to theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:14 andretains the functional activity of the NgR proteins of SEQ ID NO:2, 4 or14.

Use of mammalian host cells is expected to provide for suchpost-translational modifications (e.g., glycosylation, truncation,lipidation and phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the invention.Glycosylated and non-glycosylated forms of NgR polypeptides are embracedby the invention.

The invention also embraces variant (or analog) NgR polypeptides. In oneexample, insertion variants are provided wherein one or more amino acidresidues supplement a NgR amino acid sequence. Insertions may be locatedat either or both termini of the protein, or may be positioned withininternal regions of the NgR amino acid sequence. Insertional variantswith additional residues at either or both termini can include, forexample, fusion proteins and proteins including amino acid tags orlabels.

Insertion variants include NgR polypeptides wherein one or more aminoacid residues are added to a NgR acid sequence or to a biologicallyactive fragment thereof.

Variant products of the invention also include mature NgR products,i.e., NgR products wherein leader or signal sequences are removed, withadditional amino terminal residues. The additional amino terminalresidues may be derived from another protein, or may include one or moreresidues that are not identifiable as being derived from specificproteins. NgR products with an additional methionine residue at position−1 (Met⁻¹-NgR) are contemplated, as are variants with additionalmethionine and lysine residues at positions −2 and −1 (Met⁻²-Lys⁻¹-NgR).Variants of NgR with additional Met, Met-Lys, Lys residues (or one ormore basic residues in general) are particularly useful for enhancedrecombinant protein production in bacterial host cells.

Polypeptide Variants

The invention also embraces NgR variants having additional amino acidresidues which result from use of specific expression systems.

As used herein, a NgR “chimeric protein” or “fusion protein” comprises aNgR polypeptide operatively linked to a non-NgR polypeptide. A “NgRpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to NgR, whereas a “non-NgR polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinthat is not homologous to the NgR protein, e.g., a protein that isdifferent from the NgR protein and that is derived from the same or adifferent organism. Within a NgR fusion protein the NgR polypeptide cancorrespond to all or a portion of a NgR protein. In one embodiment, aNgR fusion protein comprises at least one biologically active portion ofa NgR protein. In another embodiment, a NgR fusion protein comprises atleast two biologically active portions of a NgR protein. In yet anotherembodiment, a NgR fusion protein comprises at least three biologicallyactive portions of a NgR protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the NgR polypeptideand the non-NgR polypeptide are fused in-frame to each other. Thenon-NgR polypeptide can be fused to the N-terminus or C-terminus of theNgR polypeptide.

For example, in one embodiment a NgR fusion protein comprises a NgRdomain operably linked to the extracellular domain of a second protein.Such fusion proteins can be further utilized in screening assays forcompounds which modulate NgR activity (such assays are described indetail below).

For example, use of commercially available vectors that express adesired polypeptide as part of a glutathione-S-transferase (GST) fusionproduct provides the desired polypeptide having an additional glycineresidue at position −1 after cleavage of the GST component from thedesired polypeptide.

In another embodiment, the fusion protein is a NgR protein containing aheterologous signal sequence at its N-terminus. For example, the nativeNgR signal sequence (i.e., amino acids 1-30 of SEQ ID NO:2 and aminoacids 1-40 of SEQ ID NO:4) can be removed and replaced with a signalsequence from another protein. In certain host cells (e.g., mammalianhost cells), expression and/or secretion NgR can be increased throughuse of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a NgR-immunoglobulinfusion protein in which the NgR sequences comprising one or more domainsare fused to sequences derived from a member of the immunoglobulinprotein family. The NgR-immunoglobulin fusion proteins of the inventioncan be incorporated into pharmaceutical compositions and administered toa subject to inhibit an interaction between NgR ligand and a NgR proteinon the surface of a cell, to thereby suppress NgR-mediated signaltransduction in vivo. NgR-immunoglobulin fusion proteins can be used toaffect the bioavailability of a NgR cognate ligand. Inhibition of theNgR ligand/NgR interaction may be useful therapeutically for both thetreatment of proliferative and differentiative disorders, as well asmodulating (e.g., promoting or inhibiting) cell survival. Moreover, theNgR-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-NgR antibodies in a subject, to purify NgRligands, and in screening assays to identify molecules that inhibit theinteraction of NgR with NgR ligand.

A NgR chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirable joiningand enzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (Eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A NgR-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theNgR protein.

Variants resulting from expression in other vector systems are alsocontemplated.

Insertional variants also include fusion proteins wherein the aminoterminus and/or the carboxy terminus of NgR is/are fused to anotherpolypeptide.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a NgR polypeptide are removed. Deletionscan be effected at one or both termini of the NgR polypeptide, or withremoval of one or more non-terminal amino acid residues of NgR. Deletionvariants, therefore, include all fragments of a NgR polypeptide.

The invention also embraces polypeptide fragments of the sequence setforth in SEQ ID NO:2, 4 or 14 wherein the fragments maintain biological(e.g., ligand binding and/or intracellular signaling) immunologicalproperties of a NgR polypeptide. Fragments comprising at least 4, 5, 10,15, 20, 25, 30, 35, or 40 consecutive amino acids of SEQ ID NO:2, 4 or14 are contemplated by the invention. Preferred polypeptide fragmentsdisplay antigenic properties unique to, or specific for, human NgR andits allelic and species homologs. Fragments of the invention having thedesired biological and immunological properties can be prepared by anyof the methods well known and routinely practiced in the art.

In still another aspect, the invention provides substitution variants ofNgR polypeptides. Substitution variants include those polypeptideswherein one or more amino acid residues of a NgR polypeptide are removedand replaced with alternative residues. In one aspect, the substitutionsare conservative in nature; however, the invention embracessubstitutions that are also non-conservative. Conservative substitutionsfor this purpose may be defined as set out in Tables 2, 3, or 4 below.

TABLE 1 X_(aa)# (based on a NTLRRCT Column I Column II domain) (R1, R2,R3) (R2 + R3 only) X₁ G, R, M X₂ A, D, C X₃ V, T X₄ N, P, S X₅ E, A, SX₆ nothing, K nothing X₇ V, M, P X₈ T, V V X₉ Q, P Q X₁₀ Q, A Q X₁₁ Q,H, N X₁₂ G, N N X₁₃ L, F F X₁₄ Q, A, S X₁₅ A, S X₁₆ V, I X₁₇ V, T, E, LX₁₈ S, G X₁₉ L, I X₂₀ A, E, V, P X₂₁ A, S, D X₂₂ S, T X₂₃ Q, E X₂₄ IVLX₂₅ Q, H Q X₂₆ N, G N X₂₇ R, L X₂₈ T, G, R, S X₂₉ F, L, T, H X₃₀ L, V LX₃₁ Q, R, P X₃₂ Q, P, A P X₃₃ G, A G X₃₄ H, T, S X₃₅ S, G, R X₃₆ P, S, AX₃₇ C, nothing nothing X₃₈ R, nothing nothing X₃₉ A, N X₄₀ M, L X₄₁ V,L, T X₄₂ T, I T X₄₃ L, I X₄₄ Y, F, H X₄₅ N, V N X₄₆ I, L X₄₇ T, S, A X₄₈F, Y, T, R X₄₉ A, H, Y, D X₅₀ P, A P X₅₁ N, S, G, A X₅₂ T, A T X₅₃ E, R,T X₅₄ G, H X₅₅ F, L X₅₆ V, Q, H X₅₇ H, A, L X₅₈ E, Q E X₅₉ G, S G X₆₀ R,A R X₆₁ Q, H Q X₆₂ R, H H X₆₃ T, S X₆₄ L, V L X₆₅ A, E, D X₆₆ E, D, AX₆₇ Q, H Q X₆₈ V, E, G X₆₉ K, R X₇₀ H, Q X₇₁ A, S, T X₇₂ Y, H X₇₃ Y, D YX₇₄ K, R X₇₅ G, Q X₇₆ S, Q S X₇₇ A, S, E X₇₈ P, G P X₇₉ A, G, P X₈₀ G, NX₈₁ I, V, L X₈₂ G, R X₈₃ H, V, A X₈₄ S, A S X₈₅ D, E X₈₆ H, S, A X₈₇ I,L X₈₈ E, L, Q X₈₉ Y, H, A X₉₀ Q, P Q X₉₁ D, N X₉₂ I, L, T X₉₃ V, A, RX₉₄ V, A, G X₉₅ S, T S X₉₆ K, R X₉₇ L, I L X₉₈ W, R, S X₉₉ S, L X₁₀₀ L,V L X₁₀₁ G, T, P X₁₀₂ Q, P, E X₁₀₃ G, H, R X₁₀₄ I, T, V, A X₁₀₅ V, G, HX₁₀₆ N, S X₁₀₇ E, G, Q X₁₀₈ Q, R X₁₀₉ L, V X₁₁₀ Q, A X₁₁₁ W, G, H X₁₁₂H, R, P X₁₁₃ K, A, H X₁₁₄ H, R X₁₁₅ D, G X₁₁₆ H, R, S, G X₁₁₇ T, M X₁₁₈T, I X₁₁₉ F, Y X₁₂₀ N, A X₁₂₁ S, N X₁₂₂ T, A, S X₁₂₃ E, S, A X₁₂₄ Q, PX₁₂₅ G, T X₁₂₆ D, E D X₁₂₇ C, A X₁₂₈ P, D X₁₂₉ V, G, P, R X₁₃₀ A, S X₁₃₁E, Q Q X₁₃₂ F, Y F X₁₃₃ G, A, D X₁₃₄ A, P X₁₃₅ D, A, V X₁₃₆ G, D X₁₃₇ A,E X₁₃₈ S, P X₁₃₉ E, A X₁₄₀ L, F X₁₄₁ R, Q X₁₄₂ R, K R X₁₄₃ R, K R X₁₄₄F, A X₁₄₅ G, V X₁₄₆ A, D, E X₁₄₇ T, P X₁₄₈ A, V, S X₁₄₉ T, S, L X₁₅₀ E,G, P, Q X₁₅₁ L, E, R X₁₅₂ R, L R X₁₅₃ G, D X₁₅₄ Q, H, A X₁₅₅ Q, R X₁₅₆K, R X₁₅₇ L, A, R X₁₅₈ R, A R X₁₅₉ V, A, E X₁₆₀ E, A, N X₁₆₁ F, L F X₁₆₂R, Q X₁₆₃ N, A, G

Variant polypeptides include those wherein conservative substitutionshave been introduced by modification of polynucleotides encodingpolypeptides of the invention. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are set out inTable 2 (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep. 6, 1996), immediately below.

TABLE 2 Conservative Substitutions I SIDE CHAIN CHARACTERISTICAMINO ACID Aliphatic Non-polar G A P I L V Polar - uncharged C S T M  N Q Polar - charged   D E   K R Aromatic H F W Y Other N Q D E

Alternatively, conservative amino acids can be grouped as described inLehninger, [BIOCHEMISTRY, Second Edition; Worth Publishers, Inc. NY,N.Y. (1975), pp. 71-77] as set out in Table 3, immediately below.

TABLE 3 Conservative Substitutions II SIDE CHAIN CHARACTERISTICAMINO ACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic:F W C. Sulfur-containing: M D. Boderline: G Uncharged-polar A. Hydroxyl:S T Y B. Amides: N Q C. Sylfhydryl: C D. Boderline: GPositively Charged (Basic): K R H Negatively Charged (Acidic):  D E

As still another alternative, exemplary conservative substitutions areset out in Table 4, below.

TABLE 4 Conservative Substitutions III Original Exemplary ResidueSubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

In addition, amino acid residues that are conserved among family membersof the NgR proteins of the present invention, as indicated by thealignment presented herein, are also predicted to be particularlyunamenable to alteration. For example, NgR proteins of the presentinvention can contain at least one domain that is a typically conservedregion in NgRs. Examples of these conserved domains include, e.g.,leucine-rich repeat domain. Amino acid residues that are not conservedor are only semi-conserved among members of the NgR proteins may bereadily amenable to alteration.

Full-length NgRs have an LRR region characterized by the amino acidconsensus sequence shown in SEQ ID NO:19. At least some full-length NgRsalso include a CT signaling (CTS) domain and a GPI domain.

The NgR domain designations used herein are defined as follows:

mNgR1 hNgR1 SEQ ID hNgR2 hNgR3 mNgR3 Domain SEQ ID: 5 NO: 17 SEQ ID: 2SEQ ID: 14 SEQ ID: 4 Signal Seq.  1-26  1-26  1-30 —  1-40 LRRNT 27-5627-56 31-59 — 41-69 LRR1 57-81 57-81 60-82  5-27 70-92 LRR2  82-105 82-105  83-106 28-51  93-106 LRR3 106-130 106-130 107-131 52-76 106-141LRR4 131-154 131-154 132-155 77-100 142-165 LRR5 155-178 155-178 156-179101-124 166-189 LRR6 179-202 179-202 180-203 125-148 190-213 LRR7203-226 203-226 204-227 149-172 214-237 LRR8 227-250 227-250 228-251173-196 238-261 LRRCT 260-309 260-309 261-310 206-255 271-320 CTS310-445 310-445 311-395 256-396 321-438 (CT Signaling) GPI 446-473456-473 396-420 370-392 439-462

In some embodiments of the invention, the above domains are modified.Modification can be in a manner that preserves domain functionality.Modification can include addition, deletion or substitution of certainamino acids. Exemplary modifications include conservative amino acidsubstitutions. Preferably such substitutions number 20 or fewer per 100residues. More preferably, such substitutions number 10 or fewer per 100residues. Further exemplary modifications include addition of flankingsequences of up to five amino acids at the N terminus and/or C terminusof one or more of the domains.

In some embodiments, the isolated nucleic acid molecule encodes apolypeptide at least about 70%, 80%, 90%, 95%, 98%, and most preferablyat least about 99% homologous to SEQ ID NO:2, 4 or 14.

Mutations can be introduced into SEQ ID NOS:1, 3 or 13 by standardtechniques, e.g., site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions can be made at one ormore amino acid residues predicted to be non-essential. Alternatively,mutations can be introduced randomly along a NgR coding sequence. Thiscan be accomplished, e.g., by saturation mutagenesis. The resultingmutants can be screened for NgR biological activity. Biologicalactivities of Ng may include but are not limited to: (1) protein:proteininteractions, e.g., with other NgRs or other cell-surface proteinsinvolved in Nogo-related signaling; (2) complex formation with a NgRligand; (3) binding to an anti-NgR antibody.

It should be understood that the definition of polypeptides of theinvention is intended to include polypeptides bearing modificationsother than insertion, deletion, or substitution of amino acid residues.By way of example, the modifications may be covalent in nature, andinclude for example, chemical bonding with polymers, lipids, otherorganic and inorganic moieties. Such derivatives may be prepared toincrease circulating half-life of a polypeptide, or may be designed toimprove the targeting capacity of the polypeptide for desired cells,tissues or organs. Similarly, the invention further embraces NgRpolypeptides that have been covalently modified to include one or morewater-soluble polymer attachments such as polyethylene glycol,polyoxyethylene glycol or polypropylene glycol. Variants that displayligand binding properties of native NgR and are expressed at higherlevels, as well as variants that provide for constitutively activereceptors, are particularly useful in assays of the invention; thevariants are also useful in providing cellular, tissue and animal modelsof diseases/conditions characterized by aberrant NgR activity.

Chemically modified NgR polypeptide compositions in which the NgRpolypeptide is linked to a polymer are included within the scope of thepresent invention. The polymer may be water soluble to preventprecipitation of the protein in an aqueous environment, such as aphysiological environment. Suitable water-soluble polymers may beselected from the group consisting of, for example, polyethylene glycol(PEG), monomethoxypolyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, polypropylene glycol homopolymers, a polypropyleneoxide/ethylene oxide copolymer polyoxyethylated polyols (e.g. glycerol)and polyvinyl alcohol. The selected polymer is usually modified to havea single reactive group, such as an active ester for acylation or analdehyde for alkylation, so that the degree of polymerization may becontrolled. Polymers may be of any molecular weight, and may be branchedor unbranched, and mixtures of such polymers may also be used. When thechemically modified NgR polymer is destined for therapeutic use,pharmaceutically acceptable polymers will be selected for use.

When the polymer is to be modified by an acylation reaction, the polymershould have a single reactive ester group. Alternatively, if the polymeris to be modified by reductive alkylation, the polymer should have asingle reactive aldehyde group. A preferred reactive aldehyde ispolyethylene glycol propionaldehyde, which is water stable, or monoCl-ClO alkoxy or aryloxy derivatives thereof (see U.S. Pat. No.5,252,714, incorporated by reference herein in its entirety).

Pegylation of NgR polypeptides may be carried out by any of thepegylation reactions known in the art, as described, for example, in thefollowing references: Focus on Growth Factors 3, 4-10 (1992); EP 0 154316; and EP 0 401 384 (each of which is incorporated by reference hereinin its entirety). Preferably, the pegylation is carried out via anacylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer). A preferred water-soluble polymer for pegylation ofpolypeptides such as NgR is polyethylene glycol (PEG). As used herein,“polyethylene glycol” is meant to encompass any of the forms of PEG thathave been used to derivatize other proteins, such as mono (Cl-ClO)alkoxy- or aryloxy-polyethylene glycol.

Chemical derivatization of NgR polypeptides may be performed under anysuitable conditions used to react a biologically active substance withan activated polymer molecule. Methods for preparing pegylated NgRpolypeptides will generally comprise the steps of (a) reacting thepolypeptide with polyethylene glycol, such as a reactive ester oraldehyde derivative of PEG, under conditions whereby NgR polypeptidebecomes attached to one or more PEG groups, and (b) obtaining thereaction products. It will be apparent to one of ordinary skill in theart to select the optimal reaction conditions or the acylation reactionsbased on known parameters and the desired result.

Pegylated and other polymer:NgR polypeptides may generally be used totreat conditions that may be alleviated or modulated by administrationof the NgR polypeptides described herein. However, thechemically-derivatized polymer:NgR polypeptide molecules disclosedherein may have additional activities, enhanced or reduced biologicalactivity, or other characteristics, such as increased or decreasedhalf-life, as compared to the nonderivatized molecules. The NgRpolypeptides, fragments thereof, variants and derivatives, may beemployed alone, together, or in combination with other pharmaceuticalcompositions. The cytokines, growth factors, antibiotics,antiinflammatories and/or chemotherapeutic agents as is appropriate forthe indication being treated.

The present invention provides compositions comprising purifiedpolypeptides of the invention. Preferred compositions comprise, inaddition to the polypeptide of the invention, a pharmaceuticallyacceptable (i.e., sterile and non-toxic) liquid, semisolid, or soliddiluent that serves as a pharmaceutical vehicle, excipient or medium.Any diluent known in the art may be used. Exemplary diluents include,but are not limited to, water, saline solutions, polyoxyethylenesorbitan monolaurate, magnesium stearate, methyl- andpropylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oiland cocoa butter.

Variants that display ligand binding properties of native NgR and areexpressed at higher levels, as well as variants that provide forconstitutively active receptors, are particularly useful in assays ofthe invention; the variants are also useful in assays of the inventionand in providing cellular, tissue and animal models ofdiseases/conditions characterized by aberrant NgR activity.

With the knowledge of the nucleotide sequence information disclosed inthe present invention, one skilled in the art can identify and obtainnucleotide sequences which encode NgR from different sources (i.e.,different tissues or different organisms) through a variety of meanswell known to the skilled artisan and as disclosed by, for example,Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which isincorporated herein by reference in its entirety.

For example, DNA that encodes NgR may be obtained by screening of mRNA,cDNA, or genomic DNA with oligonucleotide probes generated from the NgRgene sequence information provided herein. Probes may be labeled with adetectable group, such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with procedures known to theskilled artisan and used in conventional hybridization assays, asdescribed by, for example, Sambrook et al. (1989) above.

A nucleic acid molecule comprising any of the NgR nucleotide sequencesdescribed above can alternatively be synthesized by use of thepolymerase chain reaction (PCR) procedure, with the PCR oligonucleotideprimers produced from the nucleotide sequences provided herein. See U.S.Pat. Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis. The PCRreaction provides a method for selectively increasing the concentrationof a particular nucleic acid sequence even when that sequence has notbeen previously purified and is present only in a single copy in aparticular sample. The method can be used to amplify either single- ordouble-stranded DNA. The essence of the method involves the use of twooligonucleotide probes to serve as primers for the template-dependent,polymerase-mediated replication of a desired nucleic acid molecule.

A wide variety of alternative cloning and in vitro amplificationmethodologies are well known to those skilled in the art. Examples ofthese techniques are found in for example, Berger et al., Guide toMolecular Cloning Techniques, METHODS IN ENZYMOLOGY 152 Academic Press,San Diego, Calif., which is incorporated herein by reference in itsentirety.

The nucleic acid molecules of the present invention, and fragmentsderived therefrom, are useful for screening for restriction fragmentlength polymorphism (RFLP) associated with certain disorders, as well asfor genetic mapping.

Antibodies

Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, bifunctional/bispecific antibodies, humanized antibodies,human antibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a polypeptide of the invention) specific for NgRor fragments thereof. Preferred antibodies of the invention are humanantibodies which are produced and identified according to methodsdescribed in WO93/11236, published Jun. 20, 1993, which is incorporatedherein by reference in its entirety. Antibody fragments, including Fab,Fab′, F(ab′)₂, and F_(v), are also provided by the invention. The term“specific for,” when used to describe antibodies of the invention,indicates that the variable regions of the antibodies of the inventionrecognize and bind NgR polypeptides exclusively (i.e., are able todistinguish NgR polypeptides from other known NgR polypeptides by virtueof measurable differences in binding affinity, despite the possibleexistence of localized sequence identity, homology, or similaritybetween NgR and such polypeptides).

The antigenic peptide of NgR comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, 4 or 14 and encompasses anepitope of NgR such that an antibody raised against the peptide forms aspecific immune complex with NgR. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues. Preferredepitopes encompassed by the antigenic peptide are regions of NgR thatare located on the surface of the protein, e.g., hydrophilic regions.

It will be understood that specific antibodies may also interact withother proteins (for example, S. aureus protein A or other antibodies inELISA techniques) through interactions with sequences outside thevariable region of the antibodies, and, in particular, in the constantregion of the molecule. Screening assays to determine bindingspecificity of an antibody of the invention are well known and routinelypracticed in the art. For a comprehensive discussion of such assays, seeHarlow et al. in ANTIBODIES: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodiesthat recognize and bind fragments of the NgR polypeptides of theinvention are also contemplated, provided that the antibodies arespecific for NgR polypeptides. Antibodies of the invention can beproduced using any method well known and routinely practiced in the art.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of the foregoing. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed NgR protein or achemically synthesized NgR polypeptide. The preparation can furtherinclude an adjuvant. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), humanadjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum orsimilar immunostimulatory agents. If desired, the antibody moleculesdirected against NgR can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction.

The term “monoclonal antibody” or “monoclonal antibody composition,” asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of NgR. A monoclonal antibody composition thustypically displays a single binding affinity for a particular NgRprotein with which it immunoreacts. For preparation of monoclonalantibodies directed towards a particular NgR protein, or derivatives,fragments, analogs or homologs thereof, any technique that provides forthe production of antibody molecules by continuous cell line culture maybe utilized. Such techniques include, but are not limited to, thehybridoma technique (see Kohler and Milstein (1975) Nature 256,495-497); the trioma technique; the human B-cell hybridoma technique(see Kozbor et al., (1983) Immunol. Today 4, 72) and the EBV hybridomatechnique to produce human monoclonal antibodies (see Cole et al.,(1985) in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,pp. 77-96). Human monoclonal antibodies may be utilized in the practiceof the present invention and may be produced by using human hybridomas(see Cote et al., (1983) Proc Natl. Acad. Sci. USA 80, 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole etal., (1985), above).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a NgR protein (see e.g., U.S.Pat. No. 4,946,778). In addition, methods can be adapted for theconstruction of Fab expression libraries (see e.g., Huse et al., (1989)Science 246, 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a NgRprotein or derivatives, fragments, analogs or homologs thereof.Non-human antibodies can be “humanized” by techniques well known in theart. See e.g., U.S. Pat. No. 5,225,539. In one method, the non-humanCDRs are inserted into a human antibody or consensus antibody frameworksequence. Further changes can then be introduced into the antibodyframework to modulate affinity or immunogenicity. Antibody fragmentsthat contain the idiotypes to a NgR protein may be produced bytechniques known in the art including, but not limited to: (i) an F(ab)₂fragment produced by pepsin digestion of an antibody molecule; (ii) anFab fragment generated by reducing the disulfide bridges of an F(ab′)₂fragment; (iii) an Fab fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) F_(v)fragments.

Additionally, recombinant anti-NgR antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCTInternational Application No. PCT/US86/02269; European PatentApplication No. 184,187; European Patent Application No. 171,496;European Patent Application No. 173,494; PCT International PublicationNo. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent ApplicationNo. 125,023; Better et al., (1988) Science 240, 1041-1043; Liu et al.,(1987) Proc. Natl. Acad. Sci. USA 84, 3439-3443; Liu et al., (1987) J.Immunol. 139, 3521-3526; Sun et al., (1987) Proc. Natl. Acad. Sci. USA84, 214-218; Nishimura et al., (1987) Cancer Res. 47, 999-1005; Wood etal., (1985) Nature 314, 446-449; Shaw et al., (1988) J. Natl. CancerInst. 80, 1553-1559); Morrison (1985) Science 229, 1202-1207; Oi et al.,(1986) Biolechniques 4, 214; U.S. Pat. No. 5,225,539, Jones et al.,(1986) Nature 321, 552-525; Verhoeyan et al., (1988) Science 239, 1534;and Beidler et al., (1988) J. Immunol. 141, 4053-4060.

In a preferred embodiment of the invention a portion of a NgR is joinedto an Fc portion of an antibody to form a NgR/Fc fusion protein.Preferably, the Ig fusion protein is soluble. The NgR/Fc fusion proteinmay be formed by recombinant techniques as described above. In oneembodiment, a portion of a NgR including the entire amino acid sequenceof NgR except the C-terminal hydrophobic region is fused to an Fcportion of an antibody. In preferred embodiments, the NgR is a human NgRand the Fc is also human. More preferably, the human Fc portion isderived from an IgG antibody. In other embodiments, the N-terminalsignal sequence is omitted. Such antibodies are useful in binding Nogoto prevent Nogo signaling through the NgR.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of a NgR protein isfacilitated by generation of hybridomas that bind to the fragment of aNgR protein possessing such a domain. Antibodies that are specific forone or more domains within a NgR protein, e.g., domains spanning theabove-identified conserved regions of NgRs, or derivatives, fragmentsanalogs or homologs thereof, are also provided herein.

Anti-NgR antibodies may be used in methods known within the art relatingto the localization and/or quantitation of a NgR protein (e.g., for usein measuring levels of the NgR protein within appropriate physiologicalsamples, for use in diagnostic methods, for use in imaging the protein,and the like). In a given embodiment, antibodies for NgR proteins, orderivatives, fragments analogs or homologs thereof, that contain theantibody derived binding domain, are utilized aspharmacologically-active compounds [hereinafter “Therapeutics”].

An anti-NgR antibody (e.g. monoclonal antibody) can be used to isolateNgR by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-NgR antibody can facilitate thepurification of natural NgR from cells and of recombinantly produced NgRexpressed in host cells. Moreover, an anti-NgR antibody can be used todetect NgR protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of the NgRprotein. Anti-NgR antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin and aequorin, andexamples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Another aspect of the present invention is directed to methods ofinducing an immune response in a mammal against a polypeptide of theinvention by administering to the mammal an amount of the polypeptidesufficient to induce an immune response. The amount will be dependent onthe animal species, size of the animal, and the like but can bedetermined by those skilled in the art.

Another aspect of the invention is directed to anti-idiotypic antibodiesand anti-anti-idiotypic antibodies. An anti-idiotypic antibody is anantibody that recognizes determinants of another antibody (a targetantibody). Generally, the anti-idiotypic antibody recognizesdeterminants of the antigen-binding site of the target antibody.Typically, the target antibody is a monoclonal antibody. Ananti-idiotypic antibody is generally prepared by immunizing an animal(particularly, mice) of the same species and genetic type as the sourceof the target monoclonal antibody, with the target monoclonal antibody.The immunized animal mounts an immune response to the idiotypicdeterminants of the target monoclonal antibody and produces antibodiesagainst the idiotypic determinants of the target monoclonal antibody.Antibody-producing cells, such as splenic cells, of the immunized animalmay be used to generate anti-idiotypic monoclonal antibodies.Furthermore, an anti-idiotypic antibody may also be used to immunizeanimals to produce anti-anti-idiotypic antibodies. These immunizedanimals may be used to generate anti-anti-idiotypic monoclonalantibodies using standard techniques. The anti-anti-idiotypic antibodiesmay bind to the same epitope as the original, target monoclonal antibodyused to prepare the anti-idiotypic antibody. The anti-anti-idiotypicantibodies represent other monoclonal antibodies with the same antigenspecificity as the original target monoclonal antibody.

If the binding of the anti-idiotypic antibody with the target antibodyis inhibited by the relevant antigen of the target antibody, and if theanti-idiotypic antibody induces an antibody response with the samespecificity as the target antibody, it mimics the antigen of the targetantibody. Such an anti-idiotypic antibody is an “internal imageanti-idiotype” and is capable of inducing an antibody response as if itwere the original antigen. (Bona and Kohler (1984) ANTI-IDIOTYPICANTIBODIES AND INTERNAL IMAGE, IN MONOCLONAL AND ANTI-IDIOTYPICANTIBODIES: PROBES FOR RECEPTOR STRUCTURE AND FUNCTION, Venter J. C. etal. (Eds), Alan R Liss, New York, N.Y., pp 141-149, 1984) Vaccinesincorporating internal image anti-idiotype antibodies have been shown toinduce protective responses against viruses, bacteria, and parasites(Kennedy et al., (1986) 232, 220-223; 1047; McNamara et al., (1985)Science 226, 1325-1326). Internal image anti-idiotypic antibodies havealso been shown to induce immunity to tumor related antigens(Raychauhuri et al., (1986) J. Immunol. 137, 1743-1749; Raychauhuri etal., (1987) J. Immunol. 139, 3902-3910; Bhattacharya-Chatterjee et al.,(1987) J. Immunol. 139, 1354-1360; Bhattacharya-Chatterjee et al.,(1988) J. Immunol. 141, 1398-1403; Herlyn. et al. (1989) Intern. Rev.Immunol. 4, 347-357; Chen et al. (1990) Cell Imm. Immunother. Cancer351-359; Herlyn et al., (1991) in vivo 5, 615-624; Furuya et al. (1992)AntiCancer Res. 12, 27-32; Mittelman, A. et al. (1992) Proc. Natl. Acad.Sci., USA 89, 466-470; Durrant. et al., (1994) Cancer Res. 54,4837-4840; Mittelman. et al. (1994) Cancer Res. 54, 415-421; Schmitt etal. (1994) Hybridoma 13, 389-396; Chakrobarty. et al. (1995) J.Immunother. 18, 95-103; Chakrobarty. et al. (1995) Cancer Res. 55,1525-1530; Foon, K. A. et al. (1995) Clin. Cancer Res. 1, 1205-1294;Herlyn et al. (1995) Hybridoma 14, 159-166; Sclebusch et al. (1995)Hybridoma 14, 167-174; Herlyn. et al. (1996) Cancer Immunol Immunother.43, 65-76).

Anti-idiotypic antibodies for NgR may be prepared, for example, byimmunizing an animal, such as a mouse, with a immunogenic amount of acomposition comprising NgR2 (SEQ ID NO:2), NgR3 (SEQ ID NOs:4 or 14), orimmunogenic portion thereof, containing at least one antigenic epitopeof NgR. The composition may also contain a suitable adjuvant, and anycarrier necessary to provide immunogenicity. Monoclonal antibodiesrecognizing NgR may be prepared from the cells of the immunized animalas described above. A monoclonal antibody recognizing an epitope of NgRis then selected and used to prepare a composition comprising animmunogenic amount of the anti-NgR monoclonal antibody. Typically, a 25to 200 μg dose of purified anti-NgR monoclonal would be sufficient in asuitable adjuvant.

Animals may be immunized 2-6 times at 14 to 30 day intervals betweendoses. Typically, animals are immunized by any suitable route ofadministration, such as intraperitoneal, subcutaneous, intravenous or acombination of these. Anti-idiotypic antibody production may bemonitored during the immunization period using standard immunoassaymethods. Animals with suitable titers of antibodies reactive with thetarget monoclonal antibodies may be reimmunized with the monoclonalantibody used as the immunogen three days before harvesting the antibodyproducing cells. Preferably, spleen cells are used, although otherantibody producing cells may be selected. Antibody-producing cells areharvested and fused with myeloma cells to produce Hybridomas, asdescribed above, and suitable anti-idiotypic antibody-producing cellsare selected.

Anti-anti-idiotypic antibodies are produced by another round ofimmunization and Hybridoma production by using the anti-idiotypicmonoclonal antibody as the immunogen.

Antibodies of the invention are useful for, e.g., therapeutic purposes(by modulating activity of NgR), diagnostic purposes to detect orquantitate NgR, and purification of NgR. Therefore, kits comprising anantibody of the invention for any of the purposes described herein arealso comprehended.

Kits

The present invention is also directed to kits, including pharmaceuticalkits. The kits can comprise any of the nucleic acid molecules describedabove, any of the polypeptides described above, or any antibody whichbinds to a polypeptide of the invention as described above, as wellappropriate controls, such as positive and/or negative controls. The kitpreferably comprises additional components, such as, for example,instructions, solid support, reagents helpful for quantification, andthe like. For example, the kit can comprise: a labeled compound or agentcapable of detecting NgR protein or mRNA in a biological sample; meansfor determining the amount of NgR in the sample; and means for comparingthe amount of NgR in the sample with a standard. The compound or agentcan be packaged in a suitable container.

Screening Assays

The DNA and amino acid sequence information provided by the presentinvention also makes possible identification of binding partnercompounds with which a NgR polypeptide or polynucleotide will interact.Methods to identify binding partner compounds include solution assays,in vitro assays wherein NgR polypeptides are immobilized and cell-basedassays. Identification of binding partner compounds of NgR polypeptidesprovides candidates for therapeutic or prophylactic intervention inpathologies associated with NgR normal and aberrant biological activity.

The invention also provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules(e.g., molecules of less than 1,000 Daltons) or other drugs) that bindto NgR proteins or have a stimulatory or inhibitory effect on, forexample, NgR expression or NgR activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a NgRprotein or polypeptide or biologically active portion thereof. The testcompounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12,145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., (1993) Proc. Natl.Acad. Sci. USA 90, 6909; Erb et al., (1994) Proc. Natl. Acad. Sci. USA91, 11422; Zuckermann et al. (1994) J. Med. Chem. 37, 2678; Cho et al.,(1993) Science 261, 1303; Carrell et al., (1994) Angew Chem. Int. EdEngl. 33, 2059; Carell et al., (1994) Angew Chem. Int Ed Engl. 33, 2061;and Gallop et al., (1994) J. Med. Chem 37, 1233

Libraries of compounds may be presented in solution (e.g., Houghten(1992) BioTechniques. 13, 412-421), or on beads (Lam (1991) Nature 354,82-84), on chips (Fodor (1993) Nature 364, 555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner, above), plasmids (Cull et al.(1992) Proc. Natl. Acad. Sci. USA 89, 1865-1869) or on phage (Scott andSmith (1990) Science 249, 386-390; Devlin (1990) Science 249, 404-406;Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382; Felici(1991) J. Mol. Biol. 222, 301-310; Ladner, above).

1. Cell-Based Assays

The invention also provides cell-based assays to identify bindingpartner compounds of a NgR polypeptide. In one embodiment, the inventionprovides a method comprising the steps of contacting a NgR polypeptideexpressed on the surface of a cell with a candidate binding partnercompound and detecting binding of the candidate binding partner compoundto the NgR polypeptide. In another embodiment, an assay is a cell-basedassay comprising contacting a cell expressing a membrane-bound form ofNgR protein, or a biologically active portion thereof, on the cellsurface with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of theNgR protein or biologically active portion thereof.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of NgR protein, or a biologically activeportion thereof, on the cell surface is contacted with a test compoundand the ability of the test compound to bind to a NgR proteindetermined. The cell, for example, can be of mammalian origin or a yeastcell. Determining the ability of the test compound to bind to the NgRprotein can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the NgR protein or biologically active portion thereof canbe determined by detecting the labeled compound in a complex. Forexample, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemission or by scintillation counting. Alternatively,test compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product. In one embodiment, the assay comprisescontacting a cell which expresses a membrane-bound form of NgR proteinor a biologically active portion thereof, on the cell surface with aknown compound which binds NgR to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a NgR protein, wherein determining theability of the test compound to interact with a NgR protein comprisesdetermining the ability of the test compound to preferentially bind toNgR or a biologically active portion thereof as compared to the knowncompound.

Determining the ability of the test compound to modulate the activity ofNgR or a biologically active portion thereof can be accomplished, forexample, by determining the ability of the NgR protein to bind to orinteract with a NgR target molecule. As used herein, a “target molecule”is a molecule with which a NgR protein binds or interacts in nature, forexample, a molecule on the surface of a cell which expresses a NgRprotein, a molecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. A NgR target molecule can bea non-NgR molecule or a NgR protein or polypeptide of the presentinvention. In one embodiment, a NgR target molecule is a component of asignal transduction pathway that facilitates transduction of anextracellular signal (e.g., a signal generated by binding of a compoundto a membrane-bound NgR molecule) through the cell membrane and into thecell. The target, for example, can be a second intercellular proteinthat has catalytic activity or a protein that facilitates theassociation of downstream signaling molecules with NgR. In a preferredembodiment, the detection comprises detecting a calcium flux or otherphysiological event in the cell caused by the binding of the molecule.

Specific binding molecules, including natural ligands and syntheticcompounds, can be identified or developed using isolated or recombinantNgR products, NgR variants, or preferably, cells expressing suchproducts. Binding partners are useful for purifying NgR products anddetection or quantification of NgR products in fluid and tissue samplesusing known immunological procedures. Binding molecules are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) biological activities of NgR, especially those activitiesinvolved in signal transduction.

2. Cell-Free Assays

(a) Direct Binding:

The invention includes several assay systems for identifying NgR bindingpartners. In solution assays, methods of the invention comprise thesteps of (a) contacting a NgR polypeptide with one or more candidatebinding partner compounds and (b) identifying the compounds that bind tothe NgR polypeptide. Identification of the compounds that bind the NgRpolypeptide can be achieved by isolating the NgR polypeptide/bindingpartner complex and separating the binding partner compound from the NgRpolypeptide. An additional step of characterizing the physical,biological and/or biochemical properties of the binding partner compoundis also comprehended in another embodiment of the invention. In oneaspect, the NgR polypeptide/binding partner complex is isolated using anantibody immunospecific for either the NgR polypeptide or the candidatebinding partner compound.

In still other embodiments, either the NgR polypeptide or the candidatebinding partner compound comprises a label or tag that facilitates itsisolation, and methods of the invention to identify binding partnercompounds include a step of isolating the NgR polypeptide/bindingpartner complex through interaction with the label or tag. An exemplarytag of this type is a poly-histidine sequence, generally around sixhistidine residues, that permits isolation of a compound so labeledusing nickel chelation. Other labels and tags, such as the FLAG® tag(Eastman Kodak, Rochester, N.Y.), well known and routinely used in theart, are embraced by the invention.

(b) Immobilized NgR

In one variation of an in vitro assay, the invention provides a methodcomprising the steps of (a) contacting an immobilized NgR polypeptide,or a biologically active fragment thereof with a candidate bindingpartner compound and (b) detecting binding of the candidate compound tothe NgR polypeptide. In an alternative embodiment, the candidate bindingpartner compound is immobilized and binding of NgR is detected.Immobilization is accomplished using any of the methods well known inthe art, including covalent bonding to a support, a bead or achromatographic resin, as well as non-covalent, high affinityinteractions such as antibody binding, or use of streptavidin/biotinbinding wherein the immobilized compound includes a biotin moiety.Binding of a test compound to NgR, or interaction of NgR with a targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided that adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, and not by way of limitation, GST-NgRfusion proteins or GST-target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or NgR protein, and the mixture is incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, and the complexes determinedeither directly or indirectly, for example, as described above.Alternatively, the complexes can be dissociated from the matrix, and thelevel of NgR binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either NgR or itstarget molecule can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated NgR or target molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well known in theart (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with NgR or targetmolecules, but which do not interfere with binding of the NgR protein toits target molecule, can be derivatized to the wells of the plate, andunbound target or NgR trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the NgR or target molecule, aswell as enzyme-linked assays that rely on detecting an enzymaticactivity associated with the NgR or target molecule.

Detection of binding can be accomplished (i) using a radioactive labelon the compound that is not immobilized, (ii) using of a fluorescentlabel on the non-immobilized compound, (iii) using an antibodyimmunospecific for the non-immobilized compound, (iv) using a label onthe non-immobilized compound that excites a fluorescent support to whichthe immobilized compound is attached, (v) determining the activity ofthe NgR, as well as other techniques well known and routinely practicedin the art.

Determining the activity of the target molecule, for example, may beaccomplished by detecting induction of a cellular second messenger ofthe target (i.e. intracellular Ca²⁺, diacylglycerol, IP₃, etc.),detecting catalytic/enzymatic activity of the target an appropriatesubstrate, detecting the induction of a reporter gene (comprising aNgR-responsive regulatory element operatively linked to a nucleic acidencoding a detectable marker, e.g. luciferase), or detecting a cellularresponse, for example, cell survival, cellular differentiation, or cellproliferation.

(c) Competition Experiments

In yet another embodiment, the assay comprises contacting the NgRprotein or biologically active portion thereof with a known compoundwhich binds NgR to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a NgR protein, wherein determining the ability of thetest compound to interact with a NgR protein comprises determining theability of the test compound to preferentially bind to NgR orbiologically active portion thereof as compared to the known compound.

In yet another embodiment, the cell-free assay comprises contacting theNgR protein or biologically active portion thereof with a known compoundwhich binds NgR to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a NgR protein, wherein determining the ability of thetest compound to interact with a NgR protein comprises determining theability of the NgR protein to modulate the activity of a NgR targetmolecule.

The cell-free assays of the present invention are amenable to use ofboth the soluble form or the membrane-bound form of NgR. In the case ofcell-free assays comprising the membrane-bound form of NgR, it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of NgR is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS),3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

Modulators

Agents that modulate (i.e., increase, decrease, or block) NgR activityor expression may be identified by incubating a putative modulator witha cell containing a NgR polypeptide or polynucleotide and determiningthe effect of the putative modulator on NgR activity or expression. Theselectivity of a compound that modulates the activity of NgR can beevaluated by comparing its effects on NgR to its effect on other NgRcompounds. Selective modulators may include, for example, antibodies andother proteins, peptides or organic molecules which specifically bind toa NgR polypeptide or a NgR-encoding nucleic acid. Modulators of NgRactivity will be therapeutically useful in treatment of diseases andphysiological conditions in which normal or aberrant NgR activity isinvolved. NgR polynucleotides, polypeptides and modulators may be usedin the treatment of such diseases and conditions associated withdemyelination. NgR polynucleotides and polypeptides, as well as NgRmodulators, may also be used in diagnostic assays for such diseases orconditions.

Methods of the invention to identify modulators include variations onany of the methods described above to identify binding partnercompounds, the variations including techniques wherein a binding partnercompound has been identified and the binding assay is carried out in thepresence and absence of a candidate modulator. A modulator is identifiedin those instances where binding between the NgR polypeptide and thebinding partner compound changes in the presence of the candidatemodulator compared to binding in the absence of the candidate modulatorcompound. A modulator that increases binding between the NgR polypeptideand the binding partner compound is described as an enhancer oractivator, and a modulator that decreases binding between the NgRpolypeptide and the binding partner compound is described as aninhibitor.

In another embodiment, modulators of NgR expression may be identified ina method wherein a cell is contacted with a candidate compound and theexpression of NgR mRNA or protein in the cell is determined. The levelof expression of NgR mRNA or protein in the presence of the candidatecompound is compared to the level of expression of NgR mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of NgR expression based on thiscomparison. For example, when expression of NgR mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of NgR mRNA or protein expression.Alternatively, when expression of NgR mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of NgR mRNA or protein expression. The level of NgR mRNA orprotein expression in the cells can be determined by methods describedherein for detecting NgR mRNA or protein.

High Throughput Screening

The invention also comprehends high-throughput screening (HTS) assays toidentify compounds that interact with or inhibit biological activity(i.e., affect enzymatic activity, binding activity, etc.) of a NgRpolypeptide. HTS assays permit screening of large numbers of compoundsin an efficient manner. Cell-based HTS systems are contemplated toinvestigate NgR receptor-ligand interaction. HTS assays are designed toidentify “hits” or “lead compounds” having the desired property, fromwhich modifications can be designed to improve the desired property.Chemical modification of the “hit” or “lead compound” is often based onan identifiable structure/activity relationship between the “hit” andthe NgR polypeptide.

Another aspect of the present invention is directed to methods ofidentifying compounds that bind to either NgR or nucleic acid moleculesencoding NgR, comprising contacting NgR, or a nucleic acid moleculeencoding the same, with a compound, and determining whether the compoundbinds NgR or a nucleic acid molecule encoding the same. Binding can bedetermined by binding assays which are well known to the skilledartisan, including, but not limited to, gel-shift assays, Western blots,radiolabeled competition assay, phage-based expression cloning,co-fractionation by chromatography, co-precipitation, cross linking,interaction trap/two-hybrid analysis, southwestern analysis, ELISA, andthe like, which are described in, for example, Ausubel et al. (Eds.),CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1999, John Wiley & Sons, NY,which is incorporated herein by reference in its entirety. The NgRproteins, for example, can be used as “bait proteins” in a two-hybridassay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al., (1993) Cell 72, 223-232; Madura et al., (1993) J. Biol. Chem.268, 12046-12054, Bartel et al., (1993) BioTechniques 14, 920-924;Iwabuchi et al., (1993) Oncogene 8, 1693-1696; and Brent WO 94/10300),to identify other proteins that bind to or interact with NgR(“NgR-binding proteins” or “NgR-bp”) and modulate NgR activity. SuchNgR-binding proteins are also likely to be involved in the propagationof signals by the NgR proteins as, for example, upstream or downstreamelements of the NgR pathway.

Other assays may be used to identify specific ligands of a NgR receptor,including assays that identify ligands of the target protein throughmeasuring direct binding of test ligands to the target protein, as wellas assays that identify ligands of target proteins through affinityultrafiltration with ion spray mass spectroscopy/HPLC methods or otherphysical and analytical methods. Alternatively, such bindinginteractions are evaluated indirectly using the yeast two-hybrid systemdescribed in Fields et al., (1989) Nature 340, 245-246, and Fields etal., (1994) Trends Genet. 10, 286-292, both of which are incorporatedherein by reference. The two-hybrid system is a genetic assay based onthe modular nature of most transcription factors used for detectinginteractions between two proteins or polypeptides. It can be used toidentify proteins that bind to a known protein of interest, or todelineate domains or residues critical for an interaction. Variations onthis methodology have been developed to clone genes that encode DNAbinding proteins, to identify peptides that bind to a protein, and toscreen for drugs. The two-hybrid system exploits the ability of a pairof interacting proteins to bring a transcription activation domain intoclose proximity with a DNA binding domain that binds to an upstreamactivation sequence (UAS) of a reporter gene, and is generally performedin yeast. The assay requires the construction of two hybrid genesencoding (1) a DNA-binding domain that is fused to a first protein and(2) an activation domain fused to a second protein. The DNA-bindingdomain targets the first hybrid protein to the UAS of the reporter gene;however, because most proteins lack an activation domain, thisDNA-binding hybrid protein does not activate transcription of thereporter gene. The second hybrid protein, which contains the activationdomain, cannot by itself activate expression of the reporter genebecause it does not bind the UAS. However, when both hybrid proteins arepresent, the noncovalent interaction of the first and second proteinstethers the activation domain to the UAS, activating transcription ofthe reporter gene. For example, when the first protein is a NgR geneproduct, or fragment thereof, that is known to interact with anotherprotein or nucleic acid, this assay can be used to detect agents thatinterfere with the binding interaction. Expression of the reporter geneis monitored as different test agents are added to the system. Thepresence of an inhibitory agent results in lack of a reporter signal.The compounds to be screened include (which may include compounds thatare suspected to bind NgR, or a nucleic acid molecule encoding thesame), but are not limited to, extracellular, intracellular, biologicalor chemical origin.

The function of the NgR gene product is unclear and no ligands have yetbeen found which bind the gene product. The yeast two-hybrid assay isuseful to identify proteins that bind to the gene product. In an assayto identify proteins that bind to a NgR receptor, or fragment thereof, afusion polynucleotide encoding both a NgR receptor (or fragment) and aUAS binding domain (i.e., a first protein) may be used in addition, alarge number of hybrid genes each encoding a different second proteinfused to an activation domain are produced and screened in the assay.Typically, the second protein is encoded by one or more members of atotal cDNA or genomic DNA fusion library, with each secondprotein-coding region being fused to the activation domain. This systemis applicable to a wide variety of proteins, and it is not evennecessary to know the identity or function of the second bindingprotein. The system is highly sensitive and can detect interactions notrevealed by other methods; even transient interactions may triggertranscription to produce a stable mRNA that can be repeatedly translatedto yield the reporter protein.

Other assays may be used to search for agents that bind to the targetprotein. One such screening method to identify direct binding of testligands to a target protein is described in U.S. Pat. No. 5,585,277,incorporated herein by reference. This method relies on the principlethat proteins generally exist as a mixture of folded and unfoldedstates, and continually alternate between the two states. When a testligand binds to the folded form of a target protein (i.e., when the testligand is a ligand of the target protein), the target protein moleculebound by the ligand remains in its folded state. Thus, the folded targetprotein is present to a greater extent in the presence of a test ligandwhich binds the target protein, than in the absence of a ligand Bindingof the ligand to the target protein can be determined by any methodwhich distinguishes between the folded and unfolded states of the targetprotein. The function of the target protein need not be known in orderfor this assay to be performed. Virtually any agent can be assessed bythis method as a test ligand, including, but not limited to, metals,polypeptides, proteins, lipids, polysaccharides, polynucleotides andsmall organic molecules.

Another method for identifying ligands of a target protein is describedin Vieboldt et al. (1997) Anal. Chem. 69:1683-1691, incorporated hereinby reference. This technique screens combinatorial libraries of 20-30agents at a time in solution phase for binding to the target protein.Agents that bind to the target protein are separated from other librarycomponents by simple membrane washing. The specifically selectedmolecules that are retained on the filter are subsequently liberatedfrom the target protein and analyzed by HPLC and pneumatically assistedelectrospray (ion spray) ionization mass spectroscopy. This procedureselects library components with the greatest affinity for the targetprotein, and is particularly useful for small molecule libraries.

The methods of the invention also embrace ligands, especiallyneuropeptides, that are attached to a label, such as a radiolabel (e.g.,¹²⁵I, ³⁵S, ³²P, ³³P, ³H), a fluorescence label, a chemiluminescentlabel, an enzymic label and an immunogenic label. Modulators fallingwithin the scope of the invention include, but are not limited to,non-peptide molecules such as non-peptide mimetics, non-peptideallosteric effectors, and peptides. The NgR polypeptide orpolynucleotide employed in such a test may either be free in solution,attached to a solid support, borne on a cell surface or locatedintracellularly or associated with a portion of a cell. One skilled inthe art can, for example, measure the formation of complexes between NgRand the compound being tested. Alternatively, one skilled in the art canexamine the diminution in complex formation between NgR and itssubstrate caused by the compound being tested.

Another aspect of the present invention is directed to methods ofidentifying compounds which modulate (i.e., increase or decrease)activity of NgR comprising contacting NgR with a compound, anddetermining whether the compound modifies activity of NgR The activityin the presence of the test compared is measured to the activity in theabsence of the test compound. Where the activity of the samplecontaining the test compound is higher than the activity in the samplelacking the test compound, the compound will have increased activity.Similarly, where the activity of the sample containing the test compoundis lower than the activity in the sample lacking the test compound, thecompound will have inhibited activity.

The present invention is particularly useful for screening compounds byusing NgR in any of a variety of drug screening techniques. Thecompounds to be screened include (which may include compounds which aresuspected to modulate NgR activity), but are not limited to,extracellular, intracellular, biologic or chemical origin. The NgRpolypeptide employed in such a test may be in any form, preferably, freein solution, attached to a solid support, borne on a cell surface orlocated intracellularly One skilled in the art can, for example, measurethe formation of complexes between NgR and the compound being tested.Alternatively, one skilled in the art can examine the diminution incomplex formation between Nogo-R and its substrate caused by thecompound being tested.

The activity of NgR polypeptides of the invention can be determined by,for example, examining the ability to bind or be activated by chemicallysynthesized peptide ligands. Alternatively, the activity of the NgR canbe assayed by examining their ability to bind calcium ions, hormones,chemokines, neuropeptides, neurotransmitters, nucleotides, lipids,odorants and photons. Alternatively, the activity of the NgR can bedetermined by examining the activity of effector molecules including,but not limited to, adenylate cyclase, phospholipases and ion channels.Thus, modulators of NgR activity may alter a NgR receptor function, suchas a binding property of a receptor or an activity. In variousembodiments of the method, the assay may take the form of an ion fluxassay, a yeast growth assay, a non-hydrolyzable GTP assay such as a[³⁵S]-GTP S assay, a cAMP assay, an inositol triphosphate assay, adiacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPRassay for intracellular Ca²⁺ concentration, a mitogenesis assay, a MAPKinase activity assay, an arachidonic acid release assay (e.g., using[³H]-arachidonic acid) and an assay for extracellular acidificationrates, as well as other binding or function-based assays of NgR activitythat are generally known in the art. NgR activity can be determined bymethodologies that are used to assay for FaRP activity, which is wellknown to those skilled in the art. Biological activities of NgRreceptors according to the invention include, but are not limited to,the binding of a natural or an unnatural ligand, as well as any one ofthe functional activities of NgRs known in the art. Non-limitingexamples of NgR activities include transmembrane signaling of variousforms, which may involve phosphatidylinositol (PI) association and/orthe exertion of an influence over PI; another exemplary activity of NgRsis the binding of accessory proteins or polypeptides that differ fromknown GPI proteins.

The modulators of the invention exhibit a variety of chemicalstructures, which can be generally grouped into non-peptide mimetics ofnatural NgR receptor ligands, peptide and non-peptide allostericeffectors of NgR receptors, and peptides that may function as activatorsor inhibitors (competitive, uncompetitive and non-competitive) (e.g.,antibody products) of NgR receptors. The invention does not restrict thesources for suitable modulators, which may be obtained from naturalsources such as plant, animal or mineral extracts, or non-naturalsources such as small molecule libraries, including the products ofcombinatorial chemical approaches to library construction, and peptidelibraries.

Other assays can be used to examine enzymatic activity including, butnot limited to, photometric, radiometric, HPLC, electrochemical, and thelike, which are described in, for example, ENZYM ASSAYS: A PRACTICALAPPROACH, Eisenthal and Danson (Eds.), 1992, Oxford University Press,which is incorporated herein by reference in its entirety.

The use of cDNAs in drug discovery programs is well-known; assayscapable of testing thousands of unknown compounds per day inhigh-throughput screens (HTSs) are thoroughly documented. The literatureis replete with examples of the use of radiolabelled ligands in HTSbinding assays for drug discovery (see Williams (1991) Med. Res. Rev.,11, 147-184; Sweetnam et al., (1993) J. Nat. Prod 56, 441-455 forreview). Recombinant receptors are preferred for binding assay HTSbecause they allow for better specificity (higher relative purity),provide the ability to generate large amounts of receptor material, andcan be used in a broad variety of formats (see Hodgson (1992)Bio/Technology 10, 973-980; each of which is incorporated herein byreference in its entirety).

A variety of heterologous systems is available for functional expressionof recombinant receptors that are well known to those skilled in theart. Such systems include bacteria (Strosberg et al. (1992) TrendsPharmacol. Sci. 13, 95-98), yeast (Pausch (1997) Trends Biotechnol. 15,487-494), several kinds of insect cells (Vanden Broeck (1996) Int. Rev.Cytol. 164, 189-268), amphibian cells (Jayawickreme et al. (1997) Curr.Opin. Biotechnol. 8, 629-634) and several mammalian cell lines (CHO,HEK293, COS, etc.; see Gerhardt et al. (1997) Eur. J. Pharmacol. 334,1-23). These examples do not preclude the use of other possible cellexpression systems, including cell lines obtained from nematodes (PCTapplication WO 98/37177).

In preferred embodiments of the invention, methods of screening forcompounds which modulate NgR activity comprise contacting test compoundswith NgR and assaying for the presence of a complex between the compoundand NgR. In such assays, the ligand is typically labeled. After suitableincubation, free ligand is separated from that present in bound form,and the amount of free or uncomplexed label is a measure of the abilityof the particular compound to bind to NgR.

In another embodiment of the invention, high throughput screening forcompounds having suitable binding affinity to NgR is employed. Briefly,large numbers of different small peptide test compounds are synthesizedon a solid substrate. The peptide test compounds are contacted with NgRand washed. Bound NgR is then detected by methods well known in the art.Purified polypeptides of the invention can also be coated directly ontoplates for use in the aforementioned drug screening techniques. Inaddition, non-neutralizing antibodies can be used to capture the proteinand immobilize it on the solid support.

Generally, an expressed NgR can be used for HTS binding assays inconjunction with its defined ligand. The identified peptide is labeledwith a suitable radioisotope, including, but not limited to, ¹²⁵I, ³H,³⁵S or ³²P, by methods that are well known to those skilled in the art.Alternatively, the peptides may be labeled by well-known methods with asuitable fluorescent derivative (Baindur et al. (1994) Drug Dev. Res.33, 373-398; Rogers (1997) Drug Discov. Today 2, 156-160). Radioactiveligand specifically bound to the receptor in membrane preparations madefrom the cell line expressing the recombinant protein can be detected inHTS assays in one of several standard ways, including filtration of thereceptor-ligand complex to separate bound ligand from unbound ligand(Williams (1991) Med. Res. Rev. II, 147-184; Sweetnam et al. (1993) J.Nat. Prod. 56, 441-455). Alternative methods include a scintillationproximity assay (SPA) or a FlashPlate format in which such separation isunnecessary (Nakayama (1998) Curr. Opin. Drug Disc. Dev. 1, 85-91 Bosseet al. (1998) J. Biomol. Screening 3, 285-292). Binding of fluorescentligands can be detected in various ways, including fluorescence energytransfer (FRET), direct spectrophotofluorometric analysis of boundligand, or fluorescence polarization (Rogers (1997) Drug Discov. Today2, 156-160; Hill (1998) Curr. Opin. Drug Disc. Dev. 1, 92-97).

Examples of such biological responses include, but are not limited to,the following: the ability to survive in the absence of a limitingnutrient in specifically engineered yeast cells (Pausch (1997) Trends inBiotechnol. 15, 487-494); changes in intracellular Ca²⁺ concentration asmeasured by fluorescent dyes (Murphy et al. (1998) Cur. Opin Drug Disc.Dev. 1, 192-199). Fluorescence changes can also be used to monitorligand-induced changes in membrane potential or intracellular pH; anautomated system suitable for HTS has been described for these purposes(Schroeder et al. (1996) J. Biomol. Screening 1, 75-80). Melanophoresprepared from Xenopus laevis show a ligand-dependent change in pigmentorganization in response to heterologous NgR activation; this responseis adaptable to HTS formats (Jayawickreme et al. (1997) Curr. Opin.Biotechnol. 8, 629-634). Assays are also available for the measurementof common second messengers, including cAMP, phosphoinositides andarachidonic acid, but these are not generally preferred for HTS.

Preferred methods of HTS employing these receptors include permanentlytransfected CHO cells, in which agonists and antagonists can beidentified by the ability to transduce the signal for the binding ofNogo in membranes prepared from these cells through the putative GPIanchor. In another embodiment of the invention, permanently transfectedCHO cells could be used for the preparation of membranes which containsignificant amounts of the recombinant receptor proteins; these membranepreparations would then be used in receptor binding assays, employingthe radiolabelled ligand specific for the particular receptor.Alternatively, a functional assay, such as fluorescent monitoring ofligand-induced changes in internal Ca²⁺ concentration or membranepotential in permanently transfected CHO cells containing each of thesereceptors individually or in combination would be preferred for HTS.Equally preferred would be an alternative type of mammalian cell, suchas HEK293 or COS cells, in similar formats. More preferred would bepermanently transfected insect cell lines, such as Drosophila S2 cells.Even more preferred would be recombinant yeast cells expressing theDrosophila melanogaster receptors in HTS formats well known to thoseskilled in the art (e.g., Pausch (1997), above).

The invention contemplates a multitude of assays to screen and identifyinhibitors of ligand binding to NgR receptors. In one example, the NgRreceptor is immobilized and interaction with a binding partner isassessed in the presence and absence of a candidate modulator such as aninhibitor compound. In another example, interaction between the NgRreceptor and its binding partner is assessed in a solution assay, bothin the presence and absence of a candidate inhibitor compound. In eitherassay, an inhibitor is identified as a compound that decreases bindingbetween the NgR receptor and its binding partner. Another contemplatedassay involves a variation of the di-hybrid assay wherein an inhibitorof protein/protein interactions is identified by detection of a positivesignal in a transformed or transfected host cell, as described in PCTpublication number WO 95/20652, published Aug. 3, 1995.

Candidate modulators contemplated by the invention include compoundsselected from libraries of either potential activators or potentialinhibitors. There are a number of different libraries used for theidentification of small molecule modulators, including: (1) chemicallibraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules. Chemical libraries consist of random chemical structures,some of which are analogs of known compounds or analogs of compoundsthat have been identified as “hits” or “leads” in other drug discoveryscreens, some of which are derived from natural products, and some ofwhich arise from non-directed synthetic organic chemistry. Naturalproduct libraries are collections of microorganisms, animals, plants, ormarine organisms that are used to create mixtures for screening by: (1)fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of plants or marine organisms. Naturalproduct libraries include polyketides, non-ribosomal peptides, andvariants (non-naturally occurring) thereof. For a review, see Cane etal., Science (1998) 282, 63-68. Combinatorial libraries are composed oflarge numbers of peptides, oligonucleotides, or organic compounds as amixture. These libraries are relatively easy to prepare by traditionalautomated synthesis methods, PCR, cloning, or proprietary syntheticmethods. Of particular interest are non-peptide combinatorial libraries.Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries. For a review of combinatorial chemistry andlibraries created therefrom, see Myers (1997) Curr. Opin. Biotechnol. 8,701-707. Identification of modulators through use of the variouslibraries described herein permits modification of the candidate “hit”(or “lead”) to optimize the capacity of the “hit” to modulate activity.

Still other candidate inhibitors contemplated by the invention can bedesigned and include soluble forms of binding partners, as well as suchbinding partners as chimeric, or fusion, proteins. A “binding partner”as used herein broadly encompasses non-peptide modulators, as well assuch peptide modulators as neuropeptides other than natural ligands,antibodies, antibody fragments, and modified compounds comprisingantibody domains that are immunospecific for the expression product ofthe identified NgR gene.

Other embodiments of the invention comprise using competitive screeningassays in which neutralizing antibodies capable of binding a polypeptideof the invention specifically compete with a test compound for bindingto the polypeptide. In this manner, the antibodies can be used to detectthe presence of any peptide that shares one or more antigenicdeterminants with NgR. Radiolabeled competitive binding studies aredescribed in Lin et al., (1997) Antimicrob. Agents Chemother. 41,2127-2131, the disclosure of which is incorporated herein by referencein its entirety.

In other embodiments of the invention, the polypeptides of the inventionare employed as a research tool for identification, characterization andpurification of interacting, regulatory proteins. Appropriate labels areincorporated into the polypeptides of the invention by various methodsknown in the art and the polypeptides are used to capture interactingmolecules. For example, molecules are incubated with the labeledpolypeptides, washed to remove unbound polypeptides, and the polypeptidecomplex is quantified. Data obtained using different concentrations ofpolypeptide are used to calculate values for the number, affinity, andassociation of polypeptide with the protein complex.

Labeled polypeptides are also useful as reagents for the purification ofmolecules with which the polypeptide interacts including, but notlimited to, inhibitors. In one embodiment of affinity purification, apolypeptide is covalently coupled to a chromatography column. Cells andtheir membranes are extracted, and various cellular subcomponents arepassed over the column. Molecules bind to the column by virtue of theiraffinity to the polypeptide. The polypeptide-complex is recovered fromthe column, dissociated and the recovered molecule is subjected toprotein sequencing. This amino acid sequence is then used to identifythe captured molecule or to design degenerate oligonucleotides forcloning the corresponding gene from an appropriate cDNA library.

Alternatively, compounds may be identified which exhibit similarproperties to the ligand for the NgR of the invention, but which aresmaller and exhibit a longer half time than the endogenous ligand in ahuman or animal body. When an organic compound is designed, a moleculeaccording to the invention is used as a “lead” compound. The design ofmimetics to known pharmaceutically active compounds is a well-knownapproach in the development of pharmaceuticals based on such “lead”compounds. Mimetic design, synthesis and testing are generally used toavoid randomly screening a large number of molecules for a targetproperty. Furthermore, structural data deriving from the analysis of thededuced amino acid sequences encoded by the DNAs of the presentinvention are useful to design new drugs, more specific and thereforewith a higher pharmacological potency.

Comparison of the protein sequence of the present invention with thesequences present in all the available databases showed a significanthomology with the transmembrane portion of G protein coupled receptors.Accordingly, computer modeling can be used to develop a putativetertiary structure of the proteins of the invention based on theavailable information of the transmembrane domain of other proteins.Thus, novel ligands based on the predicted structure of NgR can bedesigned.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Compositions and Pharmaceutical Compositions

In a particular embodiment, the novel molecules identified by thescreening methods according to the invention are low molecular weightorganic molecules, in which case a composition or pharmaceuticalcomposition can be prepared thereof for oral or parenteraladministration. The compositions, or pharmaceutical compositions,comprising the nucleic acid molecules, vectors, polypeptides, antibodiesand compounds identified by the screening methods described herein,typically comprise the nucleic acid molecule, protein, or antibody and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The nature of the carrier or otheringredients will depend on the specific route of administration andparticular embodiment of the invention to be administered. Examples oftechniques and protocols that are useful in this context are, interalia, found in Remington's PHARMACEUTICAL SCIENCES, 16th ed., (1980)Osol, A (Ed.), which is incorporated herein by reference in itsentirety. Preferred examples of such carriers or diluents include, butare not limited to, water, saline, Ringer's solution, dextrose solutionand 5% human serum albumin. Liposomes and non-aqueous vehicles such asfixed oils may also be used. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include oral and parenteral (e.g., intravenous,intradermal, subcutaneous, inhalation, transdermal (topical),transmucosal and rectal administration). Solutions or suspensions usedfor parenteral, intradermal or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a NgR protein or anti-NgR antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811. It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91, 3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack ordispenser together with instructions for administration.

The dosage of these low molecular weight compounds will depend on thedisease state or condition to be treated and other clinical factors suchas weight and condition of the human or animal and the route ofadministration of the compound. For treating human or animals, betweenapproximately 0.5 mg/kg of body weight to 500 mg/kg of body weight ofthe compound can be administered. Therapy is typically administered atlower dosages and is continued until the desired therapeutic outcome isobserved.

Another aspect of the present invention is the use of the NgR nucleotidesequences disclosed herein for identifying homologs of the Nogo-R, inother animals, including but not limited to humans and other mammals andinvertebrates. Any of the nucleotide sequences disclosed herein, or anyportion thereof, can be used, for example, as probes to screen databasesor nucleic acid libraries, such as, for example, genomic or cDNAlibraries, to identify homologs using screening procedures well known tothose skilled in the art. Accordingly, homologs having at least 50%,more preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 95%, and most preferably at least 100% homology with NgRsequences can be identified.

The present compounds and methods, including nucleic acid molecules,polypeptides, antibodies, compounds identified by the screening methodsdescribed herein, have a variety of pharmaceutical applications and maybe used, for example, to treat or prevent unregulated cellular growth,such as cancer cell and tumor growth. In a particular embodiment, thepresent molecules are used in gene therapy. For a review of gene therapyprocedures, see e.g. Anderson Science (1992) 256, 808-813, which isincorporated herein by reference in its entirety.

The present invention also encompasses a method of agonizing(stimulating) or antagonizing a NgR natural binding partner associatedactivity in a mammal comprising administering to said mammal an agonistor antagonist to one of the above disclosed polypeptides in an amountsufficient to effect said agonism or antagonism. One embodiment of thepresent invention, then, is a method of treating diseases in a mammalwith an agonist or antagonist of the protein of the present inventioncomprising administering the agonist or antagonist to a mammal in anamount sufficient to agonize or antagonize-NgR-associated functions.

Methods of determining the dosages of compounds to be administered to apatient and modes of administering compounds to an organism aredisclosed in U.S. application Ser. No. 08/702,282, filed Aug. 23, 1996,and International patent publication number WO 96/22976, published Aug.1, 1996, both of which are incorporated herein by reference in theirentirety, including any drawings, figures or tables. Those skilled inthe art will appreciate that such descriptions are applicable to thepresent invention and can be easily adapted to it.

The proper dosage depends on various factors such as the type of diseasebeing treated, the particular composition being used and the size andphysiological condition of the patient. Therapeutically effective dosesfor the compounds described herein can be estimated initially from cellculture and animal models. For example, a dose can be formulated inanimal models to achieve a circulating concentration range thatinitially takes into account the IC₅₀ as determined in cell cultureassays. The animal model data can be used to more accurately determineuseful doses in humans.

Plasma half-life and biodistribution of the drug and metabolites in theplasma, tumors and major organs can also be determined to facilitate theselection of drugs most appropriate to inhibit a disorder. Suchmeasurements can be carried out. For example, HPLC analysis can beperformed on the plasma of animals treated with the drug and thelocation of radiolabeled compounds can be determined using detectionmethods such as X-ray, CAT scan and MRI. Compounds that show potentinhibitory activity in the screening assays, but have poorpharmacokinetic characteristics, can be optimized by altering thechemical structure and retesting. In this regard, compounds displayinggood pharmacokinetic characteristics can be used as a model.

Toxicity studies can also be carried out by measuring the blood cellcomposition. For example, toxicity studies can be carried out in asuitable animal model as follows: (1) the compound is administered tomice (an untreated control mouse should also be used); (2) blood samplesare periodically obtained via the tail vein from one mouse in eachtreatment group; and (3) the samples are analyzed for red and whiteblood cell counts, blood cell composition and the percent of lymphocytesversus polymorphonuclear cells. A comparison of results for each dosingregime with the controls indicates if toxicity is present.

At the termination of each toxicity study, further studies can becarried out by sacrificing the animals (preferably, in accordance withthe American Veterinary Medical Association guidelines Report of theAmerican Veterinary Medical Assoc. Panel on Euthanasia, (1993) J. Am.Vet Med. Assoc. 202; 229-249). Representative animals from eachtreatment group can then be examined by gross necropsy for immediateevidence of metastasis, unusual illness or toxicity. Gross abnormalitiesin tissue are noted and tissues are examined histologically. Compoundscausing a reduction in body weight or blood components are lesspreferred, as are compounds having an adverse effect on major organs. Ingeneral, the greater the adverse effect the less preferred the compound.

For the treatment of cancers the expected daily dose of a hydrophobicpharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered lessfrequently provided plasma levels of the active moiety are sufficient tomaintain therapeutic effectiveness. Plasma levels should reflect thepotency of the drug. Generally, the more potent the compound the lowerthe plasma levels necessary to achieve efficacy.

NgR mRNA transcripts have been found in the brain and heart. SEQ ID NOs:1 and/or, 3 will, as detailed above, enable screening the endogenousneurotransmitters/hormones/ligands which activate, agonize, orantagonize NgR and for compounds with potential utility in treatingdisorders including CNS disorders (e.g., stroke) and degenerativedisorders such as those associated with demyelination.

For example, NgR receptor activation may mediate the prevention ofneurite outgrowth. Inhibition would be beneficial in both chronic andacute brain injury. See, e.g., Donovan et al., (1997) J. Neurosci. 17,5316-5326; Turgeon et al., (1998) J. Neurosci. 18, 6882-6891;Smith-Swintosky et al., (1997) J. Neurochem. 69, 1890-1896; Gill et al.,(1998) Brain Res. 797, 321-327; Suidan et al., (1996) Semin. Thromb.Hemost. 22, 125-133.

Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onNgR activity (e.g., NgR gene expression), as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) disorders (e.g., a diseasecondition such as a demyelination disorder) associated with aberrant NgRactivity. In conjunction with such treatment, the pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) of theindividual may be considered. Differences in metabolism of therapeuticscan lead to severe toxicity or therapeutic failure by altering therelation between dose and blood concentration of the pharmacologicallyactive drug. Thus, the pharmacogenomics of the individual permits theselection of effective agents (e.g., drugs) for prophylactic ortherapeutic treatments based on a consideration of the individual'sgenotype. Such pharmacogenomics can further be used to determineappropriate dosages and therapeutic regimens. Accordingly, the activityof NgR protein, expression of NgR nucleic acid or mutation content ofNgR genes in an individual can be determined to thereby selectappropriate agent(s) for therapeutic or prophylactic treatment of theindividual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum (1996) Clin. Exp.Pharmacol. Physiol. 23, 983-985 and Linder (1997) Clin. Chem. 43,254-266. In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. At the other extreme are the so called ultra-rapidmetabolizers who do not respond to standard doses. Recently, themolecular basis of ultra-rapid metabolism has been identified to be dueto CYP2D6 gene amplification.

Thus, the activity of NgR protein, expression of NgR nucleic acid, ormutation content of NgR genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual. In addition, pharmacogenetic studies can beused to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a NgR modulator, such as a modulator identified by one of theexemplary screening assays described herein.

Monitoring Clinical Efficacy

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of NgR (e.g., the ability to modulate aberrantcell proliferation and/or differentiation) can be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase NgR gene expression, protein levels or upregulate NgRactivity, can be monitored in clinical trials of subjects exhibitingdecreased NgR gene expression, protein levels, or downregulated NgRactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease NgR gene expression, protein levels, ordownregulate NgR activity, can be monitored in clinical trials ofsubjects exhibiting increased NgR gene expression, protein levels, orupregulated NgR activity. In such clinical trials, the expression oractivity of NgR and, preferably, other genes that have been implicatedin, for example, a disease or disorder, can be used as a “read out” ormarkers of the immune responsiveness of a particular cell.

For example, genes, including NgR, that are modulated in cells bytreatment with an agent (e.g., compound, drug or small molecule) thatmodulates NgR activity (e.g., identified in a screening assay asdescribed herein) can be identified. Thus, to study the effect of agentson demyelination disorders, for example, in a clinical trial, cells canbe isolated and RNA prepared and analyzed for the levels of expressionof NgR and other genes implicated in the disorder. The levels of geneexpression (i.e., a gene expression pattern) can be quantified byNorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of protein produced by one of the methods asdescribed herein or by measuring the levels of activity of NgR or othergenes. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

In one embodiment, the invention provides a method for monitoring theeffectiveness of treatment of a subject with an agent (e.g., an agonist,antagonist, protein, peptide, peptidomimetic, nucleic acid, smallmolecule, or other drug candidate identified by the screening assaysdescribed herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a NgR protein, mRNA, orgenomic DNA in the preadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the NgR protein, mRNA, or genomic DNA inthe post-administration samples; (v) comparing the level of expressionor activity of the NgR protein, mRNA or genomic DNA in thepre-administration sample with the NgR protein, mRNA or genomic DNA inthe post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of NgR to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of NgR to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant NgR expression oractivity.

Diseases and disorders that are characterized by increased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that antagonize(i.e., reduce or inhibit) activity. Therapeutics that antagonizeactivity may be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, (i) aNgR polypeptide, or analogs, derivatives, fragments or homologs thereof;(ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgRpeptide; (iv) administration of antisense nucleic acid and nucleic acidsthat are “dysfunctional” (i.e., due to a heterologous insertion withinthe coding sequences of coding sequences to a NgR peptide) are utilizedto “knockout” endogenous function of a NgR peptide by homologousrecombination (see, e.g., Capecchi (1989) Science 244, 1288-1292); or(v) modulators (i.e., inhibitors, agonists and antagonists, includingadditional peptide mimetic of the invention or antibodies specific to apeptide of the invention) that alter the interaction between a NgRpeptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that increase(i.e., are agonists to) activity. Therapeutics that upregulate activitymay be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, a NgRpeptide, or analogs, derivatives, fragments or homologs thereof or anagonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of a NgRpeptide). Methods that are well-known within the art include, but arenot limited to, immunoassays (e.g., by Western blot analysis,immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, etc.).

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant NgRexpression or activity, by administering to the subject an agent thatmodulates NgR expression or at least one NgR activity. Subjects at riskfor a disease that is caused or contributed to by aberrant NgRexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the NgR aberrancy, such thata disease or disorder is prevented or, alternatively, delayed in itsprogression. Depending on the type of NgR aberrancy, for example, a NgRagonist or NgR antagonist agent can be used for treating the subject.The appropriate agent can be determined based on screening assaysdescribed herein.

Another aspect of the invention pertains to methods of modulating NgRexpression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of NgR protein activity associated withthe cell. An agent that modulates NgR protein activity can be an agentas described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a NgR protein, a peptide, a NgRpeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more NgR protein activity. Examples of suchstimulatory agents include active NgR protein and a nucleic acidmolecule encoding NgR that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more NgR protein activity.Examples of such inhibitory agents include antisense NgR nucleic acidmolecules and anti-NgR antibodies. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a NgR protein or nucleic acidmolecule. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordown-regulates) NgR expression or activity. In another embodiment, themethod involves administering a NgR protein or nucleic acid molecule astherapy to compensate for reduced or aberrant NgR expression oractivity.

Gene Therapy

Mutations in the NgR gene that result in loss of normal function of theNgR gene product underlie NgR human disease states. The inventioncomprehends gene therapy to restore NgR activity to treat those diseasestates. Delivery of a functional NgR gene to appropriate cells iseffected ex vivo, in situ, or in vivo by use of vectors, and moreparticularly viral vectors (e.g., adenovirus, adeno-associated virus, ora retrovirus), or ex vivo by use of physical DNA transfer methods (e.g.,liposomes or chemical treatments). See, for example, Anderson (1998)Nature, supplement to 392(6679):25-20. For additional reviews of genetherapy technology see Friedmann (1989) Science 244, 1275-1281; Verma(1990) Sci. Am. 68-84; and Miller (1992) Nature 357, 455-460.Alternatively, it is contemplated that in other human disease states,preventing the expression of, or inhibiting the activity of, NgR will beuseful in treating disease states. It is contemplated that antisensetherapy or gene therapy could be applied to negatively regulate theexpression of NgR.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant NgR expression oractivity.

Diseases and disorders that are characterized by increased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that antagonize(i.e., reduce or inhibit) activity. Therapeutics that antagonizeactivity may be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, (i) aNgR polypeptide, or analogs, derivatives, fragments or homologs thereof;(ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgRpeptide; (iv) administration of antisense nucleic acid and nucleic acidsthat are “dysfunctional” (i.e., due to a heterologous insertion withinthe coding sequences of coding sequences to a NgR peptide) are utilizedto “knockout” endogenous function of a NgR peptide by homologousrecombination (see, e.g., Capecchi (1989), above); or (v) modulators(i.e., inhibitors, agonists and antagonists, including additionalpeptide mimetic of the invention or antibodies specific to a peptide ofthe invention) that alter the interaction between a NgR peptide and itsbinding partner.

Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that increase(i.e., are agonists to) activity. Therapeutics that upregulate activitymay be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, a NgRpeptide, or analogs, derivatives, fragments or homologs thereof, or anagonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of a NgRpeptide) Methods that are well-known within the art include, but are notlimited to, immunoassays (e.g., by Western blot analysis,immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, etc.).

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant NgRexpression or activity, by administering to the subject an agent thatmodulates NgR expression or at least one NgR activity. Subjects at riskfor a disease that is caused or contributed to by aberrant NgRexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the NgR aberrancy, such thata disease or disorder is prevented or, alternatively, delayed in itsprogression. Depending on the type of NgR aberrancy, for example, a NgRagonist or NgR antagonist agent can be used for treating the subject.The appropriate agent can be determined based on screening assaysdescribed herein.

Another aspect of the invention pertains to methods of modulating NgRexpression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of NgR protein activity associated withthe cell. An agent that modulates NgR protein activity can be an agentas described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a NgR protein, a peptide, a NgRpeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more NgR protein activity. Examples of suchstimulatory agents include active NgR protein and a nucleic acidmolecule encoding NgR that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more NgR protein activity.Examples of such inhibitory agents include antisense NgR nucleic acidmolecules and anti-NgR antibodies. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a NgR protein or nucleic acidmolecule. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordown-regulates) NgR expression or activity. In another embodiment, themethod involves administering a NgR protein or nucleic acid molecule astherapy to compensate for reduced or aberrant NgR expression oractivity.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigure. Such modifications are intended to fall within the scope of theappended claims.

The following Table 5 contains the sequences of exemplarypolynucleotides and polypeptides of the invention.

TABLE 5 The following DNA sequence NgR2 <SEQ ID NO. 1>was identified in humans:ATGCTGCCCGGGCTCAGGCGCCTGCTGCAAGCTCCCGCCTCGGCCTGCCTCCTGCTGATGCTCCTGGCCCTGCCCCTGGCGGCCCCCAGCTGCCCCATGCTCTGCACCTGCTACTCATCCCCGCCCACCGTGAGCTGCCAGGCCAACAACTTCTCCTCTGTGCCGCTGTCCCTGCCACCCAGCACTCAGCGACTCTTCCTGCAGAACAACCTCATCCGCACGCTGCGGCCAGGCACCTTTGGGTCCAACCTGCTCACCCTGTGGCTCTTCTCCAACAACCTCTCCACCATCTACCCGGGCACTTTCCGCCACTTGCAAGCCCTGGAGGAGCTGGACCTCGGTGACAACCGGCACCTGCGCTCGCTGGAGCCCGACACCTTCCAGGGCCTGGAGCGGCTGCAGTCGCTGCATTTGTACCGCTGCCAGCTCAGCAGCCTGCCCGGCAACATCTTCCGAGGCCTGGTCAGCCTGCAGTACCTCTACCTCCAGGAGAACAGCCTGCTCCACCTACAGGATGACTTGTTCGCGGACCTGGCCAACCTGAGCCACCTCTTCCTCCACGGGAACCGCCTGCGGCTGCTCACAGAGCACGTGTTTCGCGGCCTGGGCAGCCTGGACCGGCTGCTGCTGCACGGGAACCGGCTGCAGGGCGTGCACCGCGCGGCCTTCCGCGGCCTCAGCCGCCTCACCATCCTCTACCTGTTCAACAACAGCCTGGCCTCGCTGCCCGGCGAGGCGCTCGCCGACCTGCCCTCGCTCGAGTTCCTGCGGCTCAACGCTAACCCCTGGGCGTGCGACTGCCGCGCGCGGCCGCTCTGGGCCTGGTTCCAGCGCGCGCGCGTGTCCAGCTCCGACGTGACCTGCGCCACCCCCCCGGAGCGCCAGGGCCGAGACCTGCGCGCGCTCCGCGAGGCCGACTTCCAGGCGTGTCCGCCCGCGGCACCCACGCGGCCGGGCAGCCGCGCCCGCGGCAACAGCTCCTCCAACCACCTGTACGGGGTGGCCGAGGCCGGGGCGCCCCCAGCCGATCCCTCCACCCTCTACCGAGATCTGCCTGCCGAAGACTCGCGGGGGCGCCAGGGCGGGGACGCGCCTACTGAGGACGACTACTGGGGGGGCTACGGGGGTGAGGACCAGCGAGGGGAGCAGATGTGCCCCGGCGCTGCCTGCCAGGCGCCCCCGGACTCCCGAGGCCCTGCGCTCTCGGCCGGGCTCCCCAGCCCTCTGCTTTGCCTCCTGCTCCTGGTG CCCCACCACCTCThe following amino acid sequence <SEQ ID NO. 2>is the predicted amino acid sequence derivedfrom the DNA sequence of SEQ ID NO. 1:M L P G L R R L L Q A P A S A C L L L M L L A LP L A A P S C P M L C T C Y S S P P T V S C Q AN N F S S V P L S L P P S T Q R L F L Q N N L IR T L R P G T F G S N L L T L W L F S N N L S TI Y P G T F R H L Q A L E E L D L G D N R H L RS L E P D T F Q G L E R L Q S L H L Y R C Q L SS L P G N I F R G L V S L Q Y L Y L Q E N S L LH L Q D D L F A D L A N L S H L F L H G N R L RL L T E H V F R G L G S L D R L L L H G N R L QG V H R A A F R G L S R L T I L Y L F N N S L AS L P G E A L A D L P S L E F L R L N A N P W AC D C R A R P L W A W F Q R A R V S S S D V T CA T P P E R Q G R D L R A L R E A D F Q A C P PA A P T R P G S R A R G N S S S N H L Y G V A EA G A P P A D P S T L Y R D L P A E D S R G R QG G D A P T E D D Y W G G Y G G E D Q R G E Q MC P G A A C Q A P P D S R G P A L S A G L P S P L L C L L L L V P H H LThe following DNA sequence NgR3 <SEQ ID NO. 3> was identified in mouse:ATGTCTTGGCAGTCTGGAACCACAGTGACACAATCTCCCGTGCAGGCTGCTCAGGTCTCAGGGTGCTGTGTGGAATTGCTGCTGTTGCTGCTCGCTGGAGAGCTACCTCTGGGTGGTGGTTGTCCTCGAGACTGTGTGTGCTACCCTGCGCCCATGACTGTCAGCTGCCAGGCACACAACTTTGCTGCCATCCCGGAGGGCATCCCAGAGGACAGTGAGCGCATCTTCCTGCAGAACAATCGCATCACCTTCCTCCAGCAGGGCCACTTCAGCCCCGCCATGGTCACCCTCTGGATCTACTCCAACAACATCACTTTCATTGCTCCCAACACCTTCGAGGGCTTTGTGCATCTGGAGGAGCTAGACCTTGGAGACAACCGACAGCTGCGAACGCTGGCACCCGAGACCTTCCAAGGCCTGGTGAAGCTTCACGCCCTCTACCTCTATAAGTGTGGACTGAGCGCCCTGCCCGCAGGCATCTTTGGTGGCCTGCACAGCCTGCAGTATCTCTACTTGCAGGACAACCATATCGAGTACCTCCAAGATGACATCTTTGTGGACCTGGTCAATCTCAGTCACTTGTTTCTCCATGGTAACAAGCTATGGAGCCTGGGCCAAGGCATCTTCCGGGGCCTGGTGAACCTGGACCGGTTGCTGCTGCATGAGAACCAGCTACAGTGGGTTCACCACAAGGCTTTCCATGACCTCCACAGGCTAACCACCCTCTTTCTCTTCAACAACAGCCTCACTGAGCTGCAGGGTGACTGTCTGGCCCCCCTGGTGGCCTTGGAGTTCCTTCGCCTCAATGGGAATGCTTGGGACTGTGGCTGCCGGGCACGTTCCCTGTGGGAATGGCTGCGAAGGTTCCGTGGCTCTAGCTCTGCTGTCCCCTGCGCGACCCCCGAGCTGCGGCAAGGCCAGGATCTGAAGCTGCTGAGGGTGGAGGACTTCCGGAACTGCACAGGACCAGTGTCTCCTCACCAGATCAAGTCTCACACGCTTACCACCTCTGACAGGGCTGCCCGCAAGGAGCACCATCCGTCCCATGGGGCCTCCAGGGACAAAGGCCACCCACATGGCCATCCGCCTGGCTCCAGGTCAGGTTACAAGAAGGCAGGCAAGAACTGCACCAGCCACAGGAACCGGAACCAGATCTCTAAGGTGAGCTCTGGGAAAGAGCTTACCGAACTGCAGGACTATGCCCCCGACTATCAGCACAAGTTCAGCTTTGACATCATGCCCACCGCACGACCCAAGAGGAAGGGCAAGTGTGCTCGCAGGACCCCCATCCGTGCCCCCAGTGGGGTGCAGCAGGCATCCTCAGGCACGGCCCTTGGGGCCCCACTCCTGGCCTGGATACTGGGGCTGGCAGTCACTCTCCGCThe following protein sequence <SEQ ID NO. 4> isdeduced protein of SEQ ID NO: 3:M S W Q S G T T V T Q S P V Q A A Q V S G C C VE L L L L L L A G E L P L G G G C P R D C V C YP A P M T V S C Q A H N F A A I P E G I P E D SE R I F L Q N N R I T F L Q Q G H F S P A M V TL W I Y S N N I T F I A P N T F E G F V H L E EL D L G D N R Q L R T L A P E T F Q G L V K L HA L Y L Y K C G L S A L P A G I F G G L H S L QY L Y L Q D N H I E Y L Q D D I F V D L V N L SH L F L H G N K L W S L G Q G I F R G L V N L DR L L L H E N Q L Q W V H H K A F H D L H R L TT L F L F N N S L T E L Q G D C L A P L V A L EF L R L N G N A W D C G C R A R S L W E W L R RF R G S S S A V P C A T P E L R Q G Q D L K L LR V E D F R N C T G P V S P H Q I K S H T L T TS D R A A R K E H H P S H G A S R D K G H P H GH P P G S R S G Y K K A G K N C T S H R N R N QI S K V S S G K E L T E L Q D Y A P D Y Q H K FS F D I M P T A R P K R K G K C A R R T P I R AP S G V Q Q A S S G T A L G A P L L A W I L G L A V T L RThe following protein sequence <SEQ ID NO. 5> is NgR1 from humans:M K R A S A G G S R L L A W V L W L Q A W Q V AA P C P G A C C Y N E P K V T T S C P Q Q G L QA V P V G I P A A S Q R I F L H G N R I S H V PA A S F R A C R N L T I L W L H S N A T L A R ID A A A F T G L A L L E Q L D L S D N A Q L R SV D P A T F H G L G R L H T L H L D R C G L Q EL G P G L F R G L A A L Q Y L Y L Q D N A L Q AL P D D T F R D L G N L T H L F L H G N R I S SV P E R A F R G L H S L D R L L L H Q N R V A HV H P H A F R D L G R L M T L Y L F A N N L S AL P T E A L A P L R A L Q Y L R L N D N P W V CD C R A R P L W A W L Q K F R G S S S E V P C SL P Q R L A G R D L K R L A A N D L Q G C A V AT G P Y H P I W T G R A T D E E P L G L P K C CQ P D A A D K A S V L E P G R P A S A G N A L KG R V P P G D S P P G N G S G P R H I N D S P FG T L P G S A E P P L T A V R P E G S E P P G FP T S G P R R R P G C S R K N R T R S H C R L GQ A G S G G G G T G D S E G S G A L P S L T C SL T P L G L A L V L W T V L G P CThe following amino acid sequence <SEQ ID NO: 6>is a Consensus Sequence of NgR based on homology with NgR1C P X X C X C Y X X P X X T X S C X X X X X X XX P X X X P X X X X R X F L X X N X I X X X X XX X F X X X X X X X X L W X X S N X X X X I X XX X F X X X X X L E X L D L X D N X X L R X X XP X T F X G L X X L X L X L X X C X L X X L X XX X F X G L X X L Q Y L Y L Q X N X X X X L X DD X F X D L X N L X H L F L H G N X X X X X X XX X F R G L X X L D R L L L H X N X X X X V H XX A F X X L X R L X X L X L F X N X L X X L X XX X L A X L X X L X X L R L N X N X W X C X C RA R X L W X W X X X X R X S S S X V X C X X P XX X X G X D L X X L X X X D X X X C X X X X X PX X P X X X X X X X X X X X X X X X X X X X X XX X X X X X X X X X X X X X X X G X X X X X X XX X X X X P P X X X S X X X X X X X X X X X X XX X X X X X X X X X X X X X X X X X X X X X X XX X X X X X X X X X X X X X X R X X X X X X X XX X X X X X X X X X X X X X X X X X X L X X X X X X X X X X LThe following protein sequence <SEQ ID NO: 7> isthe 66 amino acid active domain of Nogo:R I Y K G V I Q A I Q K S D E G H P F R A Y L ES E V A I S E E L V Q K Y S N S A L G H V N C TI K E L R R L F L V D D L V D S L KThe following protein sequence <SEQ ID NO: 8> isthe amino acid sequence of the mature NgR2:C P M L C T C Y S S P P T V S C Q A N N F S S VP L S L P P S T Q R L F L Q N N L I R T L R P GT F G S N L L T L W L F S N N L S T I Y P G T FR H L Q A L E E L D L G D N R H L R S L E P D TF Q G L E R L Q S L H L Y R C Q L S S L P G N IF R G L V S L Q Y L Y L Q E N S L L H L Q D D LF A D L A N L S H L F L H G N R L R L L T E H VF R G L G S L D R L L L H G N R L Q G V H R A AF R G L S R L T I L Y L F N N S L A S L P G E AL A D L P S L E F L R L N A N P W A C D C R A RP L W A W F Q R A R V S S S D V T C A T P P E RQ G R D L R A L R E A D F Q A C P P A A P T R PG S R A R G N S S S N H L Y G V A E A G A P P AD P S T L Y R D L P A E D S R G R Q G G D A P TE D D Y W G G Y G G E D Q R G E Q M C P G A A CQ A P P D S R G P A L S A G L P S P L L C L L L L V P H H LThe following protein sequence <SEQ ID NO: 9> isthe amino acid sequence of the mature NgR3:C P R D C V C Y P A P M T V S C Q A H N F A A IP E G I P E D S E R I F L Q N N R I T F L Q Q GH F S P A M V T L W I Y S N N I T F I A P N T FE G F V H L E E L D L G D N R Q L R T L A P E TF Q G L V K L H A L Y L Y K C G L S A L P A G IF G G L H S L Q Y L Y L Q D N H I E Y L Q D D IF V D L V N L S H L F L H G N K L W S L G Q G IF R G L V N L D R L L L H E N Q L Q W V H H K AF H D L H R L T T L F L F N N S L T E L Q G D CL A P L V A L E F L R L N G N A W D C G C R A RS L W E W L R R F R G S S S A V P C A T P E L RQ G Q D L K L L R V E D F R N C T G P V S P H QI K S H T L T T S D R A A R K E H H P S H G A SR D K G H P H G H P P G S R S G Y K K A G K N CT S H R N R N Q I S K V S S G K E L T E L Q D YA P D Y Q H K F S F D I M P T A R P K R K G K CA R R T P I R A P S G V Q Q A S S G T A L G A PL L A W I L G L A V T L RThe following amino acid sequence <SEQ ID NO: 10>is a conserved cysteine motif (Cysteine domain 1)of the NgR and homologs based on the Consensus Sequence:C P X X C X C Y X X P X X T X S CThe following amino acid sequence <SEQ ID NO: 11>is a conserved cysteine motif (Cysteine domain 2)of the NgR and homologs based on the Consensus Sequence:N X W X C X C R A R X L W X W X X X X R X S S SX V X C X X P X X X X G X D L X X L X X X D X X X CThe following amino acid sequence <SEQ ID NO: 12>is a conserved Leucine-rich domain of the NgR andhomologs based on the Consensus Sequence:R X F L X X N X I X X X X X X X F X X X X X X XX L W X X S N X X X X I X X X X F X X X X X L EX L D L X D N X X L R X X X P X T F X G L X X LX L X L X X C X L X X L X X X X F X G L X X L QY L Y L Q X N X X X X L X D D X F X D L X N L XH L F L H G N X X X X X X X X X F R G L X X L DR L L L H X N X X X X V H X X A F X X L X R L XX L X L F X N X L X X L X X X X L A X L X X L X X L R L

Unless otherwise indicated, X is any amino acid. For example, X whereindicated may be no amino acid. Additional features of the inventionwill be apparent from the following Examples. Examples 1-5 are actual,while the remaining Examples are prophetic.

As shown by the following Examples, a gene encoding novel NgRs have beenidentified by computational analysis of DNA sequence data. The proteinsencoded by NgR2 and NgR3 have a putative signal sequence, eightleucine-rich repeat domains in a conserved leucine-rich region (SEQ IDNO:12), a conserved cysteine-rich region (SEQ ID NO:10) N-terminal tothe leucine-rich region, a second cysteine-rich domain (SEQ ID NO:11)C-terminal to the leucine-rich region, and a putativeglycophosphatidylinositol-linkage (GPI-linkage) site. NgR2 and NgR3differ from the previously identified NgR sequence. The NgR homologs,when compared to known NgRs, show a consensus sequence (SEQ ID NOs:6).The putative mature NgR2 and NgR3 are shown in Table 5 as SEQ ID NOs: 8and 9, respectively.

Example 1 Tblastn Query of the HTG Database

The protein sequence for the human NgR (NgR1) (SEQ ID NO:5) was used toquery the high throughput genomic (HTG) database the use of which isfamiliar to those skilled in the art. The HTG database is a part ofGenBank, a comprehensive NIH genetic sequence database, which includesan annotated collection of all publicly available DNA sequences (NucleicAcids Res. (2000) 28, 15-8). The HTG database includes sequencesobtained from genomic DNA. Within genomic DNA, genes are typicallyencoded by multiple segments of DNA called exons. Thus when one aligns acDNA sequence (or a protein sequence encoded by a cDNA sequence) to agenomic sequence, the sequence will be broken up into segments dependingon the number of exons in the gene.

The BLAST algorithm, which stands for Basic Local Alignment Search Toolis suitable for determining sequence similarity (Altschul et al., (1990)J. Mol. Biol. 215, 403-410, which is incorporated herein by reference inits entirety). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The basic BLAST algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased. Extensionfor the word hits in each direction are halted when: 1) the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; 2) the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or 3)the end of either sequence is reached. The Blast algorithm parameters W,T and X determine the sensitivity and speed of the alignment. The Blastprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89,10915-10919, which is incorporated herein by reference in its entirety)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm (Karlin et al., (1993) Proc. Natl. Acad. Sci. USA90, 5873-5787, which is incorporated herein by reference) and GappedBLAST perform a statistical analysis of the similarity between twosequences. One measure of similarity provided by the BLAST algorithm isthe smallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a NgR gene or cDNA if the smallest sum probabilityin comparison of the test nucleic acid to a NgR nucleic acid is lessthan about 1, preferably less than about 0.1, more preferably less thanabout 0.01, and most preferably less than about 0.001.

To query the HTG database with the NgR protein sequence, we used avariation of the BLAST algorithm known as the tblastn program, whichcompares a protein query sequence against a nucleotide sequence databasedynamically translated in all reading frames (J. Mol. Biol. (1990) 215,403-410: Nucleic Acids Res. (1997) 25, 3389-3402). The results of thetblastn search indicated the presence of genes in the database with asignificant identity to the NgR. In addition to finding hits to genomicclones which contain the human and mouse NgR genes, we found hits toclones where the identity was not as high, but still very significant.Three human clones were found (Accession numbers: AC068514, AC016869,AC013606) with an e-value of 4e-43 and one mouse clone was found(Accession No. AC021768) with an e-value of 1e-78. The three humanclones all appeared to encode the same gene, so further analysis wasconfined to AC013606.

Example 2 Prediction of the Human NgR2 Protein Sequence (AC013606)

The human NgR protein sequence aligned with two regions of translatedsequence from nucleotide sequence AC013606, indicating that the new genewas encoded by at least two exons. In order to define the complete gene,we used the computer program GENSCAN™ (J. Mol. Biol. (1997) 268, 78-94)which can identify complete exon/intron structures of genes in genomicDNA. The gene prediction by GENESCAN™ contained seven exons. Bycomparing these predicted exons to the NgR, it was concluded that thenew human gene contains two of these exons and a part of another(containing the initiating methionine). The predicted cDNA (mRNA)encoded by these three exons was assembled from AC013606 (HTG11;deposited March 2000; length=143899; GenBank release 118.0; SEQ IDNO:15) by combining nucleotides from the three exons whose coordinatesare: 123292-123322 (exon 1); 130035-130516 (exon 2); and 138589-139335(exon 3). The sequence for this cDNA sequence is SEQ ID NO:1 (nucleotidesequence of human NgR2; AC013606). The translation of this cDNA providesthe protein sequence of human NgR2 (SEQ ID NO:2).

We used the protein sequence of human NgR2 as a query sequence againstthe human EST database. A number of hits of high significance were foundindicating that the NgR2 mRNA is expressed in a number of tissuesincluding fetal brain. Furthermore, two of these ESTs provided supportfor the exon structure that we deduced. One EST (Accession No:GB_EST19:AI346757) contains 565 nucleotides corresponding to amino acids84-271 of the human NgR2 (SEQ ID No:4). This spans the second intronlocated between amino acids 171 and 172, and provides positive evidencefor the splicing of exons 2 and 3 at the mRNA level. Another EST(GB_EST26:AI929019) contains 545 nucleotides, part of which correspondsto amino acids 1-75 of the human NgR2 (SEQ ID NO:2). This spans thefirst intron located between amino acids 10 and 11, and providespositive evidence for the splicing of exons 1 and 2 at the mRNA level.

Example 3 Prediction of the Mouse NgR3 Protein Sequence (AC021768)

The human NgR protein sequence aligned with only one region oftranslated sequence from nucleotide sequence AC021768, indicating thatmost of the new mouse gene was encoded by one large exon. However, uponinspection, the protein encoded by this exon was missing an initiatingmethionine. In order to define the complete gene, we used the computerprogram GENSCAN as described above. The gene prediction by GENSCANcontained two exons; the large one found by visual inspection and ashort one at the 5′ end which provided an initiating methionine. Thepredicted cDNA (mRNA) encoded by these two exons was assembled fromAC021768 (HTG14; deposited March 2000; length=215980; GenBank release118.0; SEQ ID NO: 16) by combining nucleotides from the two exons whosecoordinates are: the complement of 164265-164325 (exon 1); and thecomplement of 155671-156992 (exon 2). The sequence for this cDNAsequence is SEQ ID NO:3 (nucleotide sequence of mouse NgR3; AC021768).The translation of this cDNA provides the protein sequence of mouse NgR3(SEQ ID NO:4).

We used the protein sequence of mouse NgR3 as a query sequence againstthe mouse EST database. One hit of high significance was foundindicating that the NgR2 mRNA is expressed in the heart. This EST(GB_EST20:AI428334) contains 463 nucleotides, part of which correspondto amino acids 45-193 of mouse NgR3 (SEQ ID NO:4).

Example 4 Similarity Between the NgRs

An alignment between NgR1 and the two new receptors is shown in FIG.1A-1B. The similarities between these proteins include:

(1) The SignalP program, which locates the signal sequence cleavageposition, predicts a cleavage before the first conserved cysteine in allthe proteins. Thus the mature protein in all cases will have a cysteineat the N-terminus.

(2) All proteins contain eight Leucine Rich Repeats (LRR). LRRs areshort sequence motifs present in a number of proteins with diversefunctions and cellular locations. These repeats are usually involved inprotein-protein interactions. Each LRR is composed of a beta-alpha unit.

(3) All three proteins contain a leucine rich repeat N-terminal domain(LRRNT), in which four cysteines are conserved. LRRs are often flankedby cysteine rich domains at both their N and C termini.

(4) All three proteins contain a LRR C-terminal domain (LRRCT). TheLRRCTs of the three NgR proteins can be distinguished from those ofother LRR containing proteins, by the pattern of typtophans andcysteines which are completely conserved in this domain.

(5) All three proteins contain a conserved cysteine in the fourth LRRdomain.

(6) All three proteins contain a conserved potential glycosylation sitein the eighth LRR domain.

(7) NgR2 and NgR3 have a hydrophobic C-terminus, as does NGR1, anindication that they probably also undergo a modification similar toNgR1, where a GPI moiety is covalently linked to a C-terminal aminoacid. This allows the protein to remain tethered to the cell.

Example 5 Preparation of Nogo Proteins

A Nogo binding assay was developed which utilizes a method widely usedin examining semaphorin and ephrin axonal guidance function (Flanagan &Vanderhaeghen (1998) Annu. Rev. Neurosci. 21, 3 09-345, Takahashi etal., (1999) Cell 99, 59-69). It involves fusing a secreted placentalalkaline phosphatase (AP) moiety to the ligand in question to provide abiologically active receptor binding agent which can be detected with anextremely sensitive calorimetric assay. For Nogo, an expression vectoris created encoding a signal peptide, a His6 tag for purification, AP,and the 66 amino acid active domain of Nogo. The fusion protein can bepurified from the conditioned medium of transfected cells in milligramamounts. This protein is biologically active as a growth cone collapsingagent with an EC₅₀ of 1 nM.

Alternatively, a glutathione-S-transferase Nogo (GST-Nogo) fusionprotein may be prepared. For GST-Nogo, an expression vector (e.g., apGEX vector) is created encoding a signal peptide, GST, and the 66 aminoacid active domain of Nogo. GST-Nogo may be purified from the culturemedium and used as a GST fusion protein, or GST may be cleaved from theNogo portion of the fusion protein with an enzyme that recognizes thespecific amino acid cleavage sit engineered between the GST portion andthe Nogo portion of the fusion protein. Such sites are part of thecommercially available GST vectors. The specific cleavage sites andenzymes may be used in accordance with the Manufacturers specifications.

It has been found that AP-Nogo is actually slightly more potent thanGST-Nogo, perhaps because the protein is synthesized in a eukaryoticrather than a prokaryotic cell.

Binding of Nogo to immobilized NgR homologs may be performed in anELISA-type assay in which AP-Nogo is allowed to react with animmobilized receptor homolog. Specificity of binding may be demonstratedin a competitive binding assay using increasing amounts of GST-Nogo inthe type of assay to show a decreasing amount of binding of AP-Nogo (asjudged in the calorimetric assay).

Example 6 Transfected COS Cell Binding Assays

The homologs of the present invention may be used in transfectionstudies in COS cells to demonstrate binding of Nogo. Specifically,nucleotide sequences encoding NgR2 and NgR3 may be transfected into COScells using a suitable vector. Non-transfected COS-7 cells do not bindAP-Nogo. However, transfection of COS cells with nucleic acid sequencesencoding NgRs will make them capable of binding Nogo. AP alone does notbind with any stable affinity to these transfected cells, indicatingthat any affinity of Nogo for NgR2 or NgR3 would be due to the 66 aminoacids derived from Nogo. Furthermore, specific affinity of Nogo for theNgR2 or NgR3 proteins may be tested in displacement of AP-Nogo assaysusing GST-Nogo. NgR2 and/or NgR3 may also bind homologs of Nogo, whichmay also be tested using this assay.

Example 7 Expression of NgR in Human Cell Lines using Northern Blot anda Random-Primed Probe

A Northern blot is purchased from a commercial source, or RNA samplesfrom cells of interest are run on an agarose gel and blotted to amembrane using any of the well known techniques for Northern blotting.The blot is probed with a fragment of NgR2 (SEQ ID NO:1) or NgR3 (SEQ IDNO:3). The probe is prepared from 50 ng of cDNA labeled by arandom-primed method (Feinberg and Vogelstein (1983) Anal. Biochem. 132,6-13). Hybridization is carried out at 68° C. for 1 hour in ExpressHyb™solution (Clontech, Cat. No. 8015-1) followed by washing with2×SSC/0.05% SDS at room temperature and two washes with 0.1×SSC/0.1% SDSat 50° C. Expression of NgR2 and/or NgR3 can be assessed by the presenceof an appropriately sized band on the blot.

Example 8 Cloning of cDNA Corresponding to NgRs

To obtain the full-length clone corresponding to NgR2 from a cDNAlibrary, the following method may be used. A cDNA library is generatedusing standard methods from a tissue known to contain NgR2. Such atissue was identified in Example 2. 1×10⁶ plaque forming units from thecDNA library may be screened in duplicate on OPTITRAN™ filters. Thefilters are hybridized with ³²P-labeled oligonucleotides that aregenerated from the ESTs corresponding to portions of NgR2. Thehybridization reaction may consist of 400 mls plaque screen buffer (50mM Tris pH 7.5, 1M NaCl, 0.1% Sodium pyrophosphate, 0.2%Polyvinylpryolidine and 0.2% Ficoll) containing 10% Dextran sulfate and100 μg/ml tRNA and 80 μmol each ³²P-labeled oligonucleotide at 65° C.overnight. The filters are washed twice with 2×SSC/1% SDS and twice with1×SSC/1% SDS and exposed to film. Duplicate positives are purified. DNAfrom each of these clones is analyzed by restriction enzyme digestfollowed by agarose gel electrophoresis and Southern blotting. Thefilters are hybridized to the ³²P-labeled oligonucleotides used for theoriginal hybridization to confirm that inserts hybridize to the probe.The insert is then sequenced to confirm that it represents the cDNA forNgR2. Similar methods may be used to generate a full-length clonecorresponding to NgR3.

Alternatively, a full-length clone of NgR2 or NgR3 can be obtained by aperson of ordinary skill in the art employing conventional PCRtechniques.

Example 9 Hybridization Analysis to Demonstrate NgR Expression in theBrain

The expression of NgR in mammals, such as the rat, may be investigatedby in situ hybridization histochemistry. To investigate expression inthe brain, for example, coronal and sagittal rat brain cryosections (20μm thick) are prepared using a Reichert-Jung cryostat. Individualsections are thaw-mounted onto silanized, nuclease-free slides (CELAssociates, Inc., Houston, Tex.), and stored at −80° C. Sections areprocessed starting with post-fixation in cold 4% paraformaldehyde,rinsed in cold phosphate-buffered saline (PBS), acetylated using aceticanhydride in triethanolamine buffer, and dehydrated through a series ofalcohol washes in 70%, 95%, and 100% alcohol at room temperature.Subsequently, sections are delipidated in chloroform, followed byrehydration through successive exposure to 100% and 95% alcohol at roomtemperature. Microscope slides containing processed cryosections areallowed to air dry prior to hybridization. Other tissues may be assayedin a similar fashion.

A NgR-specific probe may be generated using PCR. Following PCRamplification, the fragment is digested with restriction enzymes andcloned into pBluescript II cleaved with the same enzymes. For productionof a probe specific for the sense strand of NgR, a cloned NgR fragmentcloned in pBluescript II may be linearized with a suitable restrictionenzyme, which provides a substrate for labeled run-off transcripts(i.e., cRNA riboprobes) using the vector-borne T7 promoter andcommercially available T7 RNA polymerase. A probe specific for theantisense strand of NgR may also be readily prepared using the NgR clonein pBluescript II by cleaving the recombinant plasmid with a suitablerestriction enzyme to generate a linearized substrate for the productionof labeled run-off cRNA transcripts using the T3 promoter and cognatepolymerase. The riboprobes may be labeled with [³⁵S]-UTP to yield aspecific activity of about 0.40×10⁶ cpm/pmol for antisense riboprobesand about 0.65×10⁶ cpm/pmol for sense-strand riboprobes. Each riboprobemay be subsequently denatured and added (2 pmol/ml) to hybridizationbuffer which contains 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris(pH 8.0), 1 mM EDTA, 1×Denhardt's Solution, and 10 mM dithiothreitol.Microscope slides containing sequential brain cryosections may beindependently exposed to 45 μl of hybridization solution per slide andsilanized cover slips may be placed over the sections being exposed tohybridization solution. Sections are incubated overnight (15-18 hours)at 52° C. to allow hybridization to occur. Equivalent series ofcryosections are then exposed to sense or antisense NgR-specific cRNAriboprobes.

Following the hybridization period, coverslips are washed off the slidesin 1×SSC, followed by RNase A treatment involving the exposure of slidesto 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections arethen subjected to three high-stringency washes in 0.1×SSC at 52° C. for20 minutes each. Following the series of washes, cryosections aredehydrated by consecutive exposure to 70%, 95%, and 100% ammoniumacetate in alcohol, followed by air drying and exposure to Kodak BioMax™MR-1 film. After 13 days of exposure, the film is developed, and anysignificant hybridization signal is detected. Based on these results,slides containing tissue that hybridized, as shown by filmautoradiograms, are coated with Kodak NTB-2 nuclear track emulsion andthe slides are stored in the dark for 32 days. The slides are thendeveloped and counterstained with hematoxylin. Emulsion-coated sectionsare analyzed microscopically to determine the specificity of labeling.The signal is determined to be specific if autoradiographic grains(generated by antisense probe hybridization) are clearly associated withcresyl violate-stained cell bodies. Autoradiographic grains foundbetween cell bodies indicate non-specific binding of the probe.

In some cases, such as using a probe to detect a NgR homolog in aheterologous species, in order to achieve optimal hybridization, it maybe necessary to decrease the stringency conditions. Such conditions arewell known to those of ordinary skill in the art and examples areprovided above.

Expression of NgR in the brain provides an indication that modulators ofNgR activity have utility for treating neurological disorders. Someother diseases for which modulators of NgR may have utility includedepression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia,neuropathy, neuroses, and the like. Use of NgR modulators, including NgRligands and anti-NgR antibodies, to treat individuals having suchdisease states is intended as an aspect of the invention.

Example 10 Northern Blot Analysis of NgR-RNA with a PCR-Generated Probe

Northern blot hybridizations may be performed to examine the expressionof NgR mRNA A clone containing at least a portion of the sequence of SEQID NO:1 may be used as a probe. Vector-specific primers are used in PCRto generate a hybridization probe fragment for ³²P-labeling. The PCR isperformed as follows:

Mix: 1 μl NgR-containing plasmid 2 μl fwd primer (10-50 pM) 2 μl revprimer (10-50 pM) 10 μl 10×PCR buffer (such as that provided with theenzyme, Amersham Pharmacia Biotech) 1 μl 10 mM dNTP (such as #1 969-064from Boehringer Mannheim) 0.5 μl Taq polymerase (such as #27-0799-62,Amersham Pharmacia Biotech) 83.5 μl water

PCR is performed in a Thermocycler using the following program:

94° C. 5 min 94° C. 1 min 55° C. 1 min {close oversize brace} 30 cycles72° C. 1 min 72° C. 10 min 

The PCR product may be purified using QIAquick PCR Purification Kit(#28104) from Qiagen, and radioactively labeled with ³²P-dCTP(#AA0005/250, Amersham Pharmacia Biotech)) may be done by random primingusing “Ready-to-go DNA Labeling Beads” (#27-9240-01) from AmershamPharmacia Biotech. Hybridization is carried out on Human Multiple TissueNorthern Blot from Clontech as described in manufacturer's protocol, oron a Northern Blot prepared by running RNA samples from cells ofinterest on an agarose gel and blotting to a membrane using any of theknown Northern blotting protocols. After exposure overnight on MolecularDynamics Phosphor Imager screen (#MD146-814) bands of an appropriatesize are visualized.

Example 11 Recombinant Expression of NgR in Eukaryotic Host Cells

A. Expression of NgR in Mammalian Cells

To produce NgR protein, a NgR-encoding polynucleotide is expressed in asuitable host cell using a suitable expression vector and standardgenetic engineering techniques. For example, a NgR-encoding sequencedescribed in Table 4 is subcloned into the commercial expression vectorpzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into ChineseHamster Ovary (CHO) cells using the transfection reagent FuGENE6™(Boehringer-Mannheim) and the transfection protocol provided in theproduct insert. Other eukaryotic cell lines, including human embryonickidney (HEK 293) and COS cells, are suitable as well. Cells stablyexpressing NgR are selected by growth in the presence of 100 μg/mlzeocin (Stratagene, LaJolla, Calif.). As an alternative to FuGENE6™, theexpression vector may carry the gene for dihydrofolate reductase (dhfr)and selection of clones with methotrexate (MTX) drug pressure allows forstable transformation of CHO cells. Optionally, NgR may be purified fromthe cells using standard chromatographic techniques. To facilitatepurification, antisera is raised against one or more synthetic peptidesequences that correspond to portions of the NgR amino acid sequence,and the antisera is used to affinity purify Nogo-R. The NgR also may beexpressed in-frame with a tag sequence (e.g. polyhistidine,hemaglutinin, FLAG) to facilitate purification. Moreover, it will beappreciated that many of the uses for NgR polypeptides, such as assaysdescribed below, do not require purification of NgR from the host cell:

B. Expression of NgR in CHO Cells

For expression of N in Chinese hamster ovary (CHO) cells, a plasmidbearing the relevant NgR coding sequence is prepared, using a vectorwhich also bears the selectable marker dihydrofolate reductase (DHFR).The plasmid is transfected into CHO cells. Selection under MTX drugpressure allows for preparation of stable transformants of a NgR (NgR2or NgR3) in an expression plasmid carrying a selectable marker such asDHFR.

C. Expression of NgR in 293 Cells

For expression of NgR in mammalian cells 293 (transformed human, primaryembryonic kidney cells), a plasmid bearing the relevant NgR codingsequence is prepared, using vector pSecTag2A (Invitrogen). VectorpSecTag2A contains the murine IgK chain leader sequence for secretion,the c-myc epitope for detection of the recombinant protein with theanti-myc antibody, a C-terminal polyhistidine for purification withnickel chelate chromatography, and a Zeocin resistant gene for selectionof stable transfectants. The forward primer for amplification of thisNgR cDNA is determined by routine procedures and preferably contains a5′ extension of nucleotides to introduce the HindIII cloning site andnucleotides matching the NgR sequence. The reverse primer is alsodetermined by routine procedures and preferably contains a 5′ extensionof nucleotides to introduce an XhoI restriction site for cloning andnucleotides corresponding to the reverse complement of the NgR sequence.The PCR conditions are 55° C. as the annealing temperature. The PCRproduct is gel purified and cloned into the HindIII-XhoI sites of thevector.

The DNA is purified using Qiagen chromatography columns and transfectedinto 293 cells using DOTAP™ transfection media (Boehringer Mannheim,Indianapolis, Ind.). Transiently transfected cells are tested forexpression after 24 hours of transfection, using western blots probedwith anti-His and anti-NgR peptide antibodies. Permanently transfectedcells are selected with Zeocin and propagated Production of therecombinant protein is detected from both cells and media by Westernblots probed with anti-His, anti-Myc or anti-NgR peptide antibodies.

D. Transient Expression of Nogo-R in COS Cells

For expression of the NgR in COS7 cells, a polynucleotide moleculehaving a nucleotide sequence of SEQ ID NO:1, for example, can be clonedinto vector p3-CI. This vector is a pUC18-derived plasmid that containsthe HCMV (human cytomegalovirus) promoter-intron located upstream fromthe bGH (bovine growth hormone) polyadenylation sequence and a multiplecloning site.

The forward primer is determined by routine procedures and preferablycontains a 5′ extension which introduces an XbaI restriction site forcloning, followed by nucleotides which correspond to a nucleotidesequence of SEQ ID NO:1. The reverse primer is also determined byroutine procedures and preferably contains 5′-extension of nucleotideswhich introduces a SalI cloning site followed by nucleotides whichcorrespond to the reverse complement of a nucleotide sequence of SEQ IDNO:1

The PCR consists of an initial denaturation step of 5 min at 95° C., 30cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and30 sec extension at 72° C., followed by 5 min extension at 72° C. ThePCR product is gel purified and ligated into the XbaI and SalI sites ofvector p3-CI. This construct is transformed into E. coli cells foramplification and DNA purification. The DNA is purified with Qiagenchromatography columns and transfected into COS 7 cells usingLipofectamine™ reagent from BRL, following the manufacturer's protocols.Forty-eight and 72 hours after transfection, the media and the cells aretested for recombinant protein expression.

NgR expressed from a COS cell culture can be purified by concentratingthe cell-growth media to about 10 mg of protein/ml, and purifying theprotein by, for example, chromatography. Purified NgR is concentrated to0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane andstored at −80° C. NgR3 may also be expressed using this method and thenucleotide sequence of SEQ ID NO:3 or SEQ ID NO:13.

E. Expression of NgR in Insect Cells

For expression of NgR in a baculovirus system, a polynucleotide moleculehaving a nucleotide sequence of SEQ ID NO:1, 3 or 13 can be amplified byPCR. The forward primer is determined by routine procedures andpreferably contains a 5′ extension which adds the NdeI cloning site,followed by nucleotides which correspond to a nucleotide sequence of SEQID NO:1 (or SEQ. ID NO:3 or SEQ ID NO:13, respectively). The reverseprimer is also determined by routine procedures and preferably containsa 5′ extension which introduces the KpnI cloning site, followed bynucleotides which correspond to the reverse complement of a nucleotidesequence of SEQ ID NO:1 (or SEQ ID NO:3 or SEQ ID NO:13, respectively).

The PCR product is gel purified, digested with NdeI and KpnI, and clonedinto the corresponding sites of vector pACHTL-A (Pharmingen, San Diego,Calif.). The pAcHTL expression vector contains the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV), and a 6×His tag upstream from the multiple cloning site. Aprotein kinase site for phosphorylation and a thrombin site for excisionof the recombinant protein precede the multiple cloning site is alsopresent. Of course, many other baculovirus vectors could be used inplace of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other suitablevectors for the expression of NgR polypeptides can be used, providedthat the vector construct includes appropriately located signals fortranscription, translation, and trafficking, such as an in-frame AUG anda signal peptide, as required. Such vectors are described in Luckow etal., Virology 170: 31-39, among others.

The virus is grown and isolated using standard baculovirus expressionmethods, such as those described in Summers et al. (1987) A MANUAL OFMETHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES,Texas Agricultural Experimental Station Bulletin No. 1555.

In a preferred embodiment, pAcHLT-A containing NgR gene is introducedinto baculovirus using the “BaculoGold™” transfection kit (Pharmingen,San Diego, Calif.) using methods established by the manufacturer.Individual virus isolates are analyzed for protein production byradiolabeling infected cells with ³⁵S-methionine at 24 hours postinfection Infected cells are harvested at 48 hours post infection, andthe labeled proteins are visualized by SDS-PAGE. Viruses exhibiting highexpression levels can be isolated and used for scaled up expression.

For expression of a NgR polypeptide in a Sf9 cells, a polynucleotidemolecule having the nucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO:3or SEQ ID NO:13) can be amplified by PCR using the primers and methodsdescribed above for baculovirus expression. The NgR cDNA is cloned intovector pAcHLT-A (Pharmingen) for expression in Sf9 insect. The insert iscloned into the NdeI and KpnI sites, after elimination of an internalNdeI site (using the same primers described above for expression inbaculovirus). DNA is purified with Qiagen chromatography columns andexpressed in Sf9 cells. Preliminary Western blot experiments fromnon-purified plaques are tested for the presence of the recombinantprotein of the expected size which reacted with the NgR-specificantibody. These results are confirmed after further purification andexpression optimization in HiG5 cells.

F. Expression of Soluble Forms of NgR2 and NgR3 as NgR-Ig FusionProteins.

To generate a NgR2-Ig fusion protein, standard methods may be used asdescribed in the literature (e.g. Sanicola et al. (1997) Proc. Natl.Acad. Sci. USA. 94, 6238-6243). For example, a DNA fragment encodingNgR2 without the sequence encoding the hydrophobic C-terminus (GPIanchor signal) may be ligated to a DNA fragment encoding the Fc domainof IgG1 (which may be human IgG1), and the chimeric fragment may becloned into an expression vector to generate a plasmid. The plasmid maythen be transfected into Chinese hamster ovary cells to generate astable cell line producing the fusion protein. The fusion protein isthen purified from conditioned media using standard methods. Forexample, clarified conditioned media from the cell line may be loaded bygravity directly onto Protein A Sepharose. The column may then be washedwith five column volumes each of PBS, PBS containing 0.5 M NaCl, and 25mM sodium phosphate, 100 mM NaCl (pH 5.0). The bound protein may then beeluted with 25 mM NaH₂PO₄, 100 mM NaCl (pH 2.8) and immediatelyneutralized with 1/10 fraction volume of 0.5 M Na₂HPO₄ (pH 8.6).

Similar methods may be used to generate a NgR3-Ig fusion protein.

Example 12 Interaction Trap/Two-Hybrid System

In order to assay for NgR-interacting proteins, the interactiontrap/two-hybrid library screening method can be used. This assay wasfirst described in Fields et al. (1989) Nature 340, 245, which isincorporated herein by reference in its entirety. A protocol ispublished in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1999, John Wiley &Sons, NY and Ausubel, F. M. et al. 1992, SHORT PROTOCOLS IN MOLECULARBIOLOGY, fourth edition, Greene and Wiley-interscience, NY, which isincorporated herein by reference in its entirety. Kits are availablefrom Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3).

A fusion of the nucleotide sequences encoding all or partial NgR and theyeast transcription factor GAL4 DNA-binding domain (DNA-BD) isconstructed in an appropriate plasmid (i.e., pGBKT7) using standardsubcloning techniques. Similarly, a GAL4 active domain (AD) fusionlibrary is constructed in a second plasmid (i.e. pGADT7) from cDNA ofpotential NgR-binding proteins (for protocols on forming cDNA libraries,see Sambrook et al. 1989, MOLECULAR CLONING: A LABORATORY MANUAL, secondedition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),which is incorporated herein by reference in its entirety. TheDNA-BD/NgR fusion construct is verified by sequencing, and tested forautonomous reporter gene activation and cell toxicity, both of whichwould prevent a successful two-hybrid analysis. Similar controls areperformed with the AD/library fusion construct to ensure expression inhost cells and lack of transcriptional activity. Yeast cells aretransformed (ca. 105 transformants/mg DNA) with both the NgR and libraryfusion plasmids according to standard procedure (Ausubel, et al., 1992,SHORT PROTOCOLS IN MOLECULAR BIOLOGY, fourth edition, Greene andWiley-interscience, NY, which is incorporated herein by reference in itsentirety). In vivo binding of DNA-BD/NgR with AD/library proteinsresults in transcription of specific yeast plasmid reporter genes (i.e.,lacZ, HIS3, ADE2, LEU2). Yeast cells are plated on nutrient-deficientmedia to screen for expression of reporter genes. Colonies are duallyassayed for β-galactosidase activity upon growth in Xgal(5-bromo-4-chloro-3-indolyl-b-D-galactoside) supplemented media (filterassay for b-galactosidase activity is described in Breeden et al.,(1985) Cold Spring Harb. Symp. Quant. Biol., 50, 643, which isincorporated herein by reference in its entirety). Positive AD-libraryplasmids are rescued from transformants and reintroduced into theoriginal yeast strain as well as other strains containing unrelatedDNA-BD fusion proteins to confirm specific NgR/library proteininteractions. Insert DNA is sequenced to verify the presence of an openreading frame fused to GAL4 AD and to determine the identity of theNgR-binding protein.

Example 13 Antibodies to Nogo-R

Standard techniques are employed to generate polyclonal or monoclonalantibodies to the NgR receptor, and to generate useful antigen-bindingfragments thereof or variants thereof, including “humanized” variants.Such protocols can be found, for example, in Sambrook et al. (1989),above, and Harlow et al. (Eds.), ANTIBODIES A LABORATORY MANUAL; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). In oneembodiment, recombinant NgR polypeptides (or cells or cell membranescontaining such polypeptides) are used as antigen to generate theantibodies. In another embodiment, one or more peptides having aminoacid sequences corresponding to an immunogenic portion of NgR (e.g., 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more aminoacids) are used as antigen. Peptides corresponding to extracellularportions of Nogo-R, especially hydrophilic extracellular portions, arepreferred. The antigen may be mixed with an adjuvant or linked to ahapten to increase antibody production.

A. Polyclonal or Monoclonal Antibodies

As one exemplary protocol, recombinant NgR or a synthetic fragmentthereof is used to immunize a mouse for generation of monoclonalantibodies (or larger mammal, such as a rabbit, for polyclonalantibodies). To increase antigenicity, peptides are conjugated toKeyhole Limpet Hemocyanin (Pierce), according to the manufacturer'srecommendations. For an initial injection, the antigen is emulsifiedwith Freund's Complete Adjuvant and injected subcutaneously. Atintervals of two to three weeks, additional aliquots of NgR antigen areemulsified with Freund's Incomplete Adjuvant and injectedsubcutaneously. Prior to the final booster injection, a serum sample istaken from the immunized mice and assayed by western blot to confirm thepresence of antibodies that immunoreact with NgR. Serum from theimmunized animals may be used as polyclonal antisera or used to isolatepolyclonal antibodies that recognize NgR. Alternatively, the mice aresacrificed and their spleen removed for generation of monoclonalantibodies.

To generate monoclonal antibodies, the spleens are placed in 10 mlserum-free RPMI 1640, and single cell suspensions are formed by grindingthe spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine,1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/mlstreptomycin (RPMI) (Gibco, Canada). The cell suspensions are filteredand washed by centrifugation and resuspended in serum-free RPMI.Thymocytes taken from three naive Balb/c mice are prepared in a similarmanner and used as a Feeder Layer NS-1 myeloma cells, kept in log phasein RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc.,Logan, Utah) for three days prior to fusion, are centrifuged and washedas well.

To produce hybridoma fusions, spleen cells from the immunized mice arecombined with NS-1 cells and centrifuged, and the supernatant isaspirated. The cell pellet is dislodged by tapping the tube, and 2 ml of37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) isstirred into the pellet, followed by the addition of serum-free RPMI.Thereafter, the cells are centrifuged, resuspended in RPMI containing15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine(HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×10⁶thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissueculture plates (Corning, Corning, N.Y.).

On days 2, 4, and 6 after the fusion, 100 μl of medium is removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusions are screened by ELISA, testing for the presence of mouse IgGthat binds to NgR. Selected fusion wells are further cloned by dilutionuntil monoclonal cultures producing anti-NgR antibodies are obtained.

B. Humanization of Anti-NgR Monoclonal Antibodies

The expression pattern of NgR as reported herein and the potential ofNgRs as targets for therapeutic intervention suggest therapeuticindications for NgR inhibitors (antagonists). NgR-neutralizingantibodies comprise one class of therapeutics useful as NgR antagonists.Following are protocols to improve the utility of anti-NgR monoclonalantibodies as therapeutics in humans by “humanizing” the monoclonalantibodies to improve their serum half-life and render them lessimmunogenic in human hosts (i.e., to prevent human antibody response tonon-human anti-NgR antibodies).

The principles of humanization have been described in the literature andare facilitated by the modular arrangement of antibody proteins. Tominimize the possibility of binding complement, a humanized antibody ofthe IgG4 isotype is preferred.

For example, a level of humanization is achieved by generating chimericantibodies comprising the variable domains of non-human antibodyproteins of interest with the constant domains of human antibodymolecules. (See, e.g., Morrison et al., (1989) Adv. Immunol., 44,65-92). The variable domains of NgR-neutralizing anti-NgR antibodies arecloned from the genomic DNA of a B-cell hybridoma or from cDNA generatedfrom mRNA isolated from the hybridoma of interest. The V region genefragments are linked to exons encoding human antibody constant domains,and the resultant construct is expressed in suitable mammalian hostcells (e.g., myeloma or CHO cells).

To achieve an even greater level of humanization, only those portions ofthe variable region gene fragments that encode antigen-bindingcomplementarity determining regions (“CDR”) of the non-human monoclonalantibody genes are cloned into human antibody sequences. (See, e.g.,Jones et al., (1986) Nature 321, 522-525; Riechmann et al., (1988)Nature 332, 323-327; Verhoeyen et al., (1988) Science 239, 1534-1536;and Tempest et al., (1991) Bio/Technology 9, 266-271). If necessary, theβ-sheet framework of the human antibody surrounding the CDR3 regionsalso is modified to more closely mirror the three dimensional structureof the antigen-binding domain of the original monoclonal antibody. (SeeKettleborough et al., (1991) Protein Engin. 4, 773-783; and Foote etal., (1992) J. Mol. Biol. 224, 487-499).

In an alternative approach, the surface of a non-human monoclonalantibody of interest is humanized by altering selected surface residuesof the non-human antibody, e.g., by site-directed mutagenesis, whileretaining all of the interior and contacting residues of the non-humanantibody. See Padlan (1991) Mol. Immunol. 28, 489-498.

The foregoing approaches are employed using NgR-neutralizing anti-NgRmonoclonal antibodies and the hybridomas that produce them to generatehumanized NgR-neutralizing antibodies useful as therapeutics to treat orpalliate conditions wherein NgR expression or ligand-mediated NgRsignaling is detrimental.

C. Human NgR-Neutralizing Antibodies from Phage Display

Human NgR-neutralizing antibodies are generated by phage displaytechniques such as those described in Aujame et al. (1997) HumanAntibodies 8, 155-168; Hoogenboom (1997) TIBTECH 15, 62-70; and Rader etal. (1997), Curr. Opin. Biotechnol. 8, 503-508, all of which areincorporated by reference. For example, antibody variable regions in theform of Fab fragments or linked single chain Fv fragments are fused tothe amino terminus of filamentous phage minor coat protein pIII.Expression of the fusion protein and incorporation thereof into themature phage coat results in phage particles that present an antibody ontheir surface and contain the genetic material encoding the antibody. Aphage library comprising such constructs is expressed in bacteria, andthe library is screened for NgR-specific phage-antibodies using labeledor immobilized NgR as antigen-probe.

D. Human NgR-Neutralizing Antibodies from Transgenic Mice

Human NgR-neutralizing antibodies are generated in transgenic miceessentially as described in Bruggemann et al. (1996) Immunol. Today 17,391-397 and Bruggemann et al. (1997) Curr. Opin. Biotechnol. 8, 455-458.Transgenic mice carrying human V-gene segments in germline configurationand that express these transgenes in their lymphoid tissue are immunizedwith a NgR composition using conventional immunization protocolshybridomas are generated using B cells from the immunized mice usingconventional protocols and screened to identify hybridomas secretinganti-NgR human antibodies (e.g., as described above).

Example 14 Assays to Identify Modulators of NgR Activity

Set forth below are several nonlimiting assays for identifyingmodulators (agonists and antagonists) of NgR activity. Among themodulators that can be identified by these assays are natural ligandcompounds of the receptor; synthetic analogs and derivatives of naturalligands; antibodies, antibody fragments, and/or antibody-like compoundsderived from natural antibodies or from antibody-like combinatoriallibraries; and/or synthetic compounds identified by high-throughputscreening of libraries; and the like. All modulators that bind NgR areuseful for identifying NgR in tissue samples (e.g., for diagnosticpurposes, pathological purposes, and the like). Agonist and antagonistmodulators are useful for up-regulating and down-regulating NgRactivity, respectively, to treat disease states characterized byabnormal levels of NgR activity. The assays may be performed usingsingle putative modulators, and/or may be performed using a knownagonist in combination with candidate antagonists (or visa versa).

A. cAMP Assays

In one type of assay, levels of cyclic adenosine monophosphate (cAMP)are measured in NgR-transfected cells that have been exposed tocandidate modulator compounds. Protocols for cAMP assays have beendescribed in the literature. (See, e.g., Sutherland et al., (1968)Circulation 37, 279; Frandsen et al., (1976) Life Sciences 18, 529-541;Dooley et al., (1997) J. Pharmacol. Exp. Therap. 283, 735-41; and Georgeet al., (1997) J. Biomol. Screening 2, 235-40). An exemplary protocolfor such an assay, using an Adenylyl Cyclase Activation FlashPlate®Assay from NEN™ Life Science Products, is set forth below.

Briefly, the NgR coding sequence (e.g., a cDNA or intronless genomicDNA) is subcloned into a commercial expression vector, such as pzeoSV2(Invitrogen), and transiently transfected into Chinese Hamster Ovary(CHO) cells using known methods, such as the transfection protocolprovided by Boehringer-Mannheim when supplying the FuGENE 6 transfectionreagent. Transfected CHO cells are seeded into 96-well microplates fromthe FlashPlate® assay kit, which are coated with solid scintillant towhich antisera to cAMP has been bound. For a control, some wells areseeded with wild type (untransfected) CHO cells. Other wells in theplate receive various amounts of a cAMP standard solution for use increating a standard curve.

One or more test compounds (i.e., candidate modulators) are added to thecells in each well, with water and/or compound-free medium/diluentserving as a control or controls. After treatment, cAMP is allowed toaccumulate in the cells for exactly 15 minutes at room temperature. Theassay is terminated by the addition of lysis buffer containing[¹²⁵I]-labeled cAMP, and the plate is counted using a Packard Topcount™96-well microplate scintillation counter. Unlabeled cAMP from the lysedcells (or from standards) and fixed amounts of [¹²⁵I]-cAMP compete forantibody bound to the plate. A standard curve is constructed, and cAMPvalues for the unknowns are obtained by interpolation. Changes inintracellular cAMP levels of cells in response to exposure to a testcompound are indicative of NgR modulating activity. Modulators that actas agonists of receptors which couple to the G_(s) subtype of G proteinswill stimulate production of cAMP, leading to a measurable 3-10 foldincrease in cAMP levels. Agonists of receptors which couple to theG_(i/o) subtype of G proteins will inhibit forskolin-stimulated cAMPproduction, leading to a measurable decrease in cAMP levels of 50-100%.Modulators that act as inverse agonists will reverse these effects atreceptors that are either constitutively active or activated by knownagonists.

B. Aequorin Assays

In another assay, cells (e.g., CHO cells) are transiently co-transfectedwith both a NgR expression construct and a construct that encodes thephotoprotein apoaquorin. In the presence of the cofactor coelenterazine,apoaquorin will emit a measurable luminescence that is proportional tothe amount of intracellular (cytoplasmic) free calcium. (See generally,Cobbold, et al. “Aequorin measurements of cytoplasmic free calcium,” In:McCormack J. G. and Cobbold P. H., eds., CELLULAR CALCIUM: A PRACTICALAPPROACH. Oxford:IRL Press (1991); Stables et al., (1997) Anal. Biochem.252, 115-26; and Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCHCHEMICALS. Sixth edition. Molecular Probes, Eugene, Oreg. (1996)).

In one exemplary assay, NgR is subcloned into the commercial expressionvector pzeoSV2 (Invitrogen) and transiently co-transfected along with aconstruct that encodes the photoprotein apoaquorin (Molecular Probes,Eugene, Oreg.) into CHO cells using the transfection reagent FuGENE 6(Boehringer-Mannheim) and the transfection protocol provided in theproduct insert.

The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL,Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 2 mMglutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at which timethe medium is changed to serum-free MEM containing 5 μM coelenterazine(Molecular Probes, Eugene, Oreg.). Culturing is then continued for twoadditional hours at 37° C. Subsequently, cells are detached from theplate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000cells/ml in serum-free MEM.

Dilutions of candidate NgR modulator compounds are prepared inserum-free MEM and dispensed into wells of an opaque 96-well assay plateat 50 μl/well. Plates are then loaded onto an MLX microtiter plateluminometer (Dynex Technologies, Inc., Chantilly, Va.). The instrumentis programmed to dispense 50 μl cell suspensions into each well, onewell at a time, and immediately read luminescence for 15 seconds.Dose-response curves for the candidate modulators are constructed usingthe area under the curve for each light signal peak. Data are analyzedwith SlideWrite, using the equation for a one-site ligand, and EC₅₀values are obtained. Changes in luminescence caused by the compounds areconsidered indicative of modulatory activity. Modulators that act asagonists at receptors which couple to the Gq subtype of G proteins givean increase in luminescence of up to 100 fold. Modulators that act asinverse agonists will reverse this effect at receptors that are eitherconstitutively active or activated by known agonists.

C. Luciferase Reporter Gene Assay

The photoprotein luciferase provides another useful tool for assayingfor modulators of NgR activity. Cells (e.g., CHO cells or COS 7 cells)are transiently co-transfected with both a NgR expression construct(e.g., NgR in pzeoSV2) and a reporter construct which includes a genefor the luciferase protein downstream from a transcription factorbinding site, such as the cAMP-response element (CRE), AP-1, or NF-kappaB. Expression levels of luciferase reflect the activation status of thesignaling events. (See generally, George et al. (1997) J. Biomol.Screening 2, 235-240; and Stratowa et al. (1995) Curr. Opin. Biotechnol.6, 574-581). Luciferase activity may be quantitatively measured using,e.g., luciferase assay reagents that are commercially available fromPromega (Madison, Wis.).

In one exemplary assay, CHO cells are plated in 24-well culture dishesat a density of 100,000 cells/well one day prior to transfection andcultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovineserum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.Cells are transiently co-transfected with both a NgR expressionconstruct and a reporter construct containing the luciferase gene. Thereporter plasmids CRE-luciferase, AP-1-luciferase andNF-kappaB-luciferase may be purchased from Stratagene (Legally, Calif.).Transfections are performed using the FuGENE 6 transfection reagent(Boehringer-Mannheim) according to the supplier's instructions. Cellstransfected with the reporter construct alone are used as a control.Twenty-four hours after transfection, cells are washed once with PBSpre-warmed to 37° C. Serum-free MEM is then added to the cells eitheralone (control) or with one or more candidate modulators and the cellsare incubated at 37° C. for five hours. Thereafter, cells are washedonce with ice-cold PBS and lysed by the addition of 100 μl of lysisbuffer per well from the luciferase assay kit supplied by Promega. Afterincubation for 15 minutes at room temperature, 15 μl of the lysate ismixed with 50 μl of substrate solution (Promega) in an opaque-white,96-well plate, and the luminescence is read immediately on a Wallacemodel 1450 MicroBeta scintillation and luminescence counter (WallaceInstruments, Gaithersburg, Md.).

Differences in luminescence in the presence versus the absence of acandidate modulator compound are indicative of modulatory activity.Receptors that are either constitutively active or activated by agoniststypically give a 3-20-fold stimulation of luminescence compared to cellstransfected with the reporter gene alone. Modulators that act as inverseagonists will reverse this effect.

D. Intracellular Calcium Measurement using FLIPR

Changes in intracellular calcium levels are another recognized indicatorof receptor activity, and such assays can be employed to screen formodulators of NgR activity. For example, CHO cells stably transfectedwith a NgR expression vector are plated at a density of 4×10⁴ cells/wellin Packard black-walled, 96-well plates specially designed todiscriminate fluorescence signals emanating from the various wells onthe plate. The cells are incubated for 60 minutes at 37° C. in modifiedDulbecco's PBS (D-PBS) containing 36 mg/L pyruvate and 1 g/L glucosewith the addition of 1% fetal bovine serum and one of four calciumindicator dyes (Fluo-3™ AM, Fluo-4™ AM, Calcium Green™-1 AM, or OregonGreen™ 488 BAPTA-1 AM), each at a concentration of 4 μM. Plates arewashed once with modified D-PBS without 1% fetal bovine serum andincubated for 10 minutes at 37° C. to remove residual dye from thecellular membrane. In addition, a series of washes with modified D-PBSwithout 1% fetal bovine serum is performed immediately prior toactivation of the calcium response.

A calcium response is initiated by the addition of one or more candidatereceptor agonist compounds, calcium ionophore A23187 (10 μM; positivecontrol), or ATP (4 μM; positive control). Fluorescence is measured byMolecular Device's FLIPR with an argon laser (excitation at 488 nm).(See, e.g., Kuntzweiler et al. (1998) Drug Dev. Res. 44, 14-20). TheF-stop for the detector camera is set at 2.5 and the length of exposureis 0.4 milliseconds. Basal fluorescence of cells is measured for 20seconds prior to addition of candidate agonist, ATP, or A23187, and thebasal fluorescence level is subtracted from the response signal. Thecalcium signal is measured for approximately 200 seconds, takingreadings every two seconds. Calcium ionophore A23187 and ATP increasethe calcium signal 200% above baseline levels. In general, activatedNgRs increase the calcium signal at least about 10-15% above baselinesignal.

E. [³⁵S]GTPγS Binding Assay

It is also possible to evaluate whether NgR signals through a Gprotein-mediated pathway. Because G protein-coupled receptors signalthrough intracellular G proteins whose activity involves GTP binding andhydrolysis to yield bound GDP, measurement of binding of thenon-hydrolyzable GTP analog [³⁵S]-GTPγS in the presence and absence ofcandidate modulators provides another assay for modulator activity.(See, e.g., Kowal et al., (1998) Neuropharmacology 37, 179-187.).

In one exemplary assay, cells stably transfected with a NgR expressionvector are grown in 10 cm tissue culture dishes to subconfluence, rinsedonce with 5 ml of ice-cold Ca²⁺/Mg²⁺-free phosphate-buffered saline, andscraped into 5 ml of the same buffer. Cells are pelleted bycentrifugation (500×g, 5 minutes), resuspended in TEE buffer (25 mMTris, pH 7.5, 5 mM EDTA, 5 mM EGTA), and frozen in liquid nitrogen.After thawing, the cells are homogenized using a Dounce homogenizer (1ml TEE per plate of cells), and centrifuged at 1,000×g for 5 minutes toremove nuclei and unbroken cells.

The homogenate supernatant is centrifuged at 20,000×g for 20 minutes toisolate the membrane fraction, and the membrane pellet is washed oncewith TEE and resuspended in binding buffer (20 mM BEPES, pH 7.5, 150 mMNaCl, 10 mM MgCl₂, 1 mM EDTA). The resuspended membranes can be frozenin liquid nitrogen and stored at −70° C. until use.

Aliquots of cell membranes prepared as described above and stored at−70° C. are thawed, homogenized, and diluted into buffer containing 20mM HEPES, 10 mM MgCl₂, 1 mM EDTA, 120 mM NaCl, 10 μM GDP, and 0.2 mMascorbate, at a concentration of 10-50 μg/ml. In a final volume of 90μl, homogenates are incubated with varying concentrations of candidatemodulator compounds or 100 μM GTP for 30 minutes at 30° C. and thenplaced on ice. To each sample, 10 μl guanosine 5′-O-(3-[³⁵S]thio)triphosphate (NEN, 1200 Cimmol; [³⁵S]-GTPγS), was added to a finalconcentration of 100-200 μM. Samples are incubated at 30° C. for anadditional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl₂, at 4°C. is added and the reaction is stopped by filtration.

Samples are filtered over Whatman GF/B filters and the filters arewashed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl₂. Filters arecounted by liquid scintillation spectroscopy. Nonspecific binding of[³⁵]-GTPγS is measured in the presence of 100 μM GTP and subtracted fromthe total. Compounds are selected that modulate the amount of[³⁵S]-GTPγS binding in the cells, compared to untransfected controlcells. Activation of receptors by agonists gives up to a five-foldincrease in [³⁵S]-GTPγS binding This response is blocked by antagonists.

F. [³H]Arachidonic Acid Release

The activation of NgRs may also potentiate arachidonic acid release incells, providing yet another useful assay for modulators of NgRactivity. (See, e.g., Kanterman et al., (1991) Mol. Pharmacol. 39,364-369.) For example, CHO cells that are stably transfected with a NgRexpression vector are plated in 24-well plates at a density of 15,000cells/well and grown in MEM medium supplemented with 10% fetal bovineserum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin for48 hours at 37° C. before use. Cells of each well are labeled byincubation with [³H]-arachidonic acid. (Amersham Corp., 210 Ci/mmol) at0.5 μCi/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5%fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells arethen washed twice with 1 ml of the same buffer.

Candidate modulator compounds are added in 1 ml of the same buffer,either alone or with 10 μM ATP and the cells are incubated at 37° C. for30 minutes. Buffer alone and mock-transfected cells are used ascontrols. Samples (0.5 ml) from each well are counted by liquidscintillation spectroscopy. Agonists which activate the receptor willlead to potentiation of the ATP-stimulated release of [³H]-arachidonicacid. This potentiation is blocked by antagonists.

G. Extracellular Acidification Rate

In yet another assay, the effects of candidate modulators of NgRactivity are assayed by monitoring extracellular changes in pH inducedby the test compounds (see, e.g., Dunlop et al. (1998) J. Pharmacol.Toxicol. Meth 40, 47-55). In one embodiment, CHO cells transfected witha NgR expression vector are seeded into 12 mm capsule cups (MolecularDevices Corp.) at 4×10⁵ cells/cup in MEM supplemented with 10% fetalbovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/mlstreptomycin. The cells are incubated in this medium at 37° C. in 5% CO₂for 24 hours.

Extracellular acidification rates are measured using a Cytosensormicrophysiometer (Molecular Devices Corp.). The capsule cups are loadedinto the sensor chambers of the microphysiometer and the chambers areperfused with running buffer (bicarbonate-free MEM supplemented with 4mM L-glutamine, 10 units/ml penicillin, 10 μg/ml streptomycin, 26 mMNaCl) at a flow rate of 100 μl/minute. Candidate agonists or otheragents are diluted into the running buffer and perfused through a secondfluid path. During each 60-second pump cycle, the pump is run for 38seconds and is off for the remaining 22 seconds. The pH of the runningbuffer in the sensor chamber is recorded during the cycle from 43-58seconds, and the pump is re-started at 60 seconds to start the nextcycle. The rate of acidification of the running buffer during therecording time is calculated by the Cytosoft program. Changes in therate of acidification are calculated by subtracting the baseline value(the average of 4 rate measurements immediately before addition of amodulator candidate) from the highest rate measurement obtained afteraddition of a modulator candidate. The selected instrument detects 61mV/pH unit. Modulators that act as agonists of the receptor result in anincrease in the rate of extracellular acidification compared to the ratein the absence of agonist. This response is blocked by modulators whichact as antagonists of the receptor.

Example 15 mNgR3 does not Bind hNogo-A(1055-1120)

To functionally test the mouse NgR3 (hereinafter, mNgR3) for its abilityto bind hNogo-A(1055-1120), a cDNA expression vector for a mycepitope-tagged mNgR3 protein was created. The mouse NgR3 cDNA wasamplified by PCR from mouse adult brain cDNA, from the signal sequenceto the stop codon, and ligated into the pSecTag2 vector such that thevector encodes a signal sequence followed by a myc tag followed by themature mNgR3 sequence. This plasmid was transfected into COS07 cells,and expression of a myc-tagged protein of the predicted size wasverified by immunoblot analysis. Alkaline phosphatase-hNogo-A(1055-1120)binding studies and myc immunohistology were conducted as described(Fournier et al., supra).

The cells expressing mNgR3 express the myc-tagged protein but binding toAP-hNogo-A(1055-1120) was not observed under the conditions employed(FIG. 8).

Example 16 Identification of Partial Human NgR3 cDNA and ProteinSequences

The tblastn program was used to search for the human homolog of mouseNgR3. The mouse NgR3 protein sequence (SEQ ID NO:4) was used to query aproprietary human expressed sequence tag (EST) database from Incyteyielding one highly significant hit: Incyte Template ID 190989.1. Thissequence (937 nucleotides) contains an open reading frame of 312 aminoacids in the second reverse frame that exhibits 88% identity withresidues 66 to 381 of mouse NgR3 (SEQ ID NO:4), strongly indicating thatit is part of the human NgR3 homolog.

A query of SEQ ID NO:4 against the public human EST database in Genbankalso produced a hit with a 465-bp EST (Accession number: R35699; Versionnumber: R35699.1; GI: 792600). There are a number of single nucleotidedeletions and insertions within this sequence which cause frame shifterrors. All of the reliable sequence contained in this public EST ispresent in the Incyte EST (Template ID 190989.1).

To obtain more nucleotide sequence that would extend the amino acidsequence at that carboxy terminal end, the I.M.A.G.E. Consortium cloneNo. 38319, which corresponds to Genbank accession No. R35699, waspurchased from Incyte Genomics Inc. and subjected to further DNAsequence analysis. This clone consists of a NotI/HinD III fragmentcontaining the sequence of interest, cloned into the Not/HinD III sitesof the vector Lafinid BA(http://image.llnl.gov/image/html/libs/lafmidBA.shtml). The clone wasreceived as an agar stab, which was streaked out on LB agar platescontaining 50 ug/ml ampicillin to isolate individual colonies. Sixcolonies were grown in LB medium with antibiotic, and plasmid DNA wasprepared using the Promega Wizard Plus Miniprep DNA Purification System(Promega #A7500). These DNAs were subsequently digested with NotI andHinD III restriction enzymes to confirm that the clones contained aninsert. The insert of one isolate was sequenced using a combination ofvector specific and gene specific primers yielding a partial nucleotidesequence of human NgR3 of 1176 nucleotides (SEQ ID NO:13). A translationof this sequence provides a partial sequence for human NgR3 of 392 aminoacids (SEQ ID NO:14).

The nucleotide sequence of SEQ ID NO:13 differs from the Incyte ESTsequence at three positions. Nucleotide positions 12-13 in SEQ ID NO:13are CG, whereas the corresponding nucleotides in the Incyte Template ID190989.1 are GT (i.e., positions 12-13 of the complement of IncyteTemplate ID 190989.1). In addition, position 641 in SEQ ID NO:13 is a C,whereas the corresponding nucleotide in the Incyte Template ID 190989.1sequence is an A (i.e., position 641 of the complement of IncyteTemplate ID 190989.1). This results in two changes in amino acids whencomparing SEQ ID NO:14 to the ORF encoded by Incyte Template 190989.1:SEQ ID NO:14 contains a valine at position 5, whereas the ORF encoded byIncyte Template ID 190989.1 contains a leucine; SEQ ID NO:14 contains analanine at position 214, whereas the ORF encoded by Incyte Template ID190989.1 contains a glutamic acid.

The nucleotide sequence of SEQ ID NO:13 differs from the public EST(Accession number: R35699; Version number: R35699.1; GI: 792600)sequence at two positions (within the first 200 nucleotides of reliablesequence). Nucleotide positions 12-13 in SEQ ID NO:13 are CG, whereasthe corresponding nucleotides in the public EST are GT (i.e., positions12-13 of the public EST; Accession no: R135699; Version no: R35699.1;GI: 792600) This leads to a single amino acid change when comparing SEQID NO:14 to the ORF encoded by the public EST: SEQ ID NO:14 contains avaline at position 5, while the ORF encoded by the public EST contains aleucine.

A Bestfit analysis of the partial human amino acid sequence with thefull-length mouse amino acid sequence indicates that the human NgR3amino acid sequence is complete at the carboxy terminal end and thatthey share 89.54% identity. An alignment of all the NgR proteins isshown in FIG. 9. Although the human NgR3 amino acid sequence is missingthe first 25 amino acids, it can be determined that the human NgR3protein contains the following features in common with the other NgRsequences: (1) eight Leucine Rich Repeat (LRR) domains; (2) an LRRcarboxy-terminal (LRR-CT) domain; (3) a conserved cysteine in the fourthLRR domain; (4) a conserved potential glycosylation site in the eighthLRR domain; and (5) a hydrophobic carboxyl terminus.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

The entire disclosure of each publication cited herein is herebyincorporated by reference. This application claims benefit from U.S.provisional application 60/238,361, filed Oct. 6, 2000, which isincorporated by reference herein in its entirety.

KEY FOR SEQUENCE LISTING

-   SEQ ID NO:1 human NgR2 cDNA sequence derived from genomic sequence    AC013606-   SEQ ID NO:2 human NgR2 amino acid sequence-   SEQ ID NO:3 mouse NgR3 cDNA sequence derived from AC021768-   SEQ ID NO:4 a mouse NgR3 amino acid sequence-   SEQ ID NO:5 a human NgR1 amino acid sequence-   SEQ ID NO:6 a consensus amino acid sequence for NgRs-   SEQ ID NO:7 #1055-1120 amino acid residues of hNogoA (Nogo-66)-   SEQ ID NO:8 a mature human NgR2 amino acid sequence-   SEQ ID NO:9 a mature mouse NgR3 amino acid sequence-   SEQ ID NO:10 a consensus NgR LLRNT amino acid sequence-   SEQ ID NO:11 a consensus NgR LRRCT domain amino acid sequence-   SEQ ID NO:12 a consensus NgR LRR domain amino acid sequence-   SEQ ID NO:13 a partial human NgR3 nucleotide sequence-   SEQ ID NO:14 a partial human NgR3 amino acid sequence-   SEQ ID NO:15 a genomic sequence encoding a human NgR2 sequence.-   SEQ ID NO:16 a genomic sequence (complementary strand) encoding a    mouse NgR3-   SEQ ID NO:17 a mouse NgR1 amino acid sequence-   SEQ ID NO:18 a consensus sequence for the NTLRRCT domain of NgR-   SEQ ID NO:19 an consensus NgR LRRCT domain amino acid sequence

1-3. (canceled)
 4. An isolated nucleic acid encoding the polypeptide of SEQ ID NO:
 2. 5-16. (canceled)
 17. An isolated polypeptide comprising an amino sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:14. 18-22. (canceled)
 23. An isolated antibody that binds to the polypeptide of claim
 17. 24-25. (canceled)
 26. A method of decreasing inhibition of axonal growth of a CNS neuron, comprising the step of contacting the neuron with an effective amount of the polypeptide of claim
 17. 27. A method of treating a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of the polypeptide of claim
 17. 28. A method of decreasing inhibition of axonal growth of a CNS neuron comprising the step of contacting the neuron with an effective amount of the antibody according to claim
 23. 29. A method of treating a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of the antibody according to claim
 23. 30. A method for identifying a molecule that binds the polypeptide of claim 17 comprising: (a) providing the polypeptide of claim 17; (b) contacting the polypeptide with a candidate molecule; (c) detecting binding of the candidate molecule to said polypeptide. 